LANA viral protein binding sites

ABSTRACT

This invention relates to novel reagents and methods for the screening of compounds that may be agonistic or antagonistic to the binding of viral DNA binding proteins to viral DNA. The invention also relates to methods that may be used in gene therapy and that may be used to treat tumor viruses.

[0001] This invention was made in part with government support from apublic service grant NCI CA72150-01. The government has certain rightsin the invention.

FIELD OF THE INVENTION

[0002] This invention generally relates to novel compounds and methodsfor the detection of compounds that are agonistic or antagonistic forthe binding of viral genetic material to genomic host DNA. Additionally,the inventions generally relates to compounds and methods related togene transfer and gene therapy, as well as therapeutics for virallybased diseases.

BACKGROUND

[0003] In 1872, Moritz Kaposi described a multifocal vascular tumoraffecting elderly men of Mediterranean or Eastern European origin. Morerecently, this neoplasm has become prevalent in immunocompromisedpatients, such as transplant recipients on immunosuppressive therapy,and AIDS patients, where it has become the most common cancer (Beral, V.“Epidemiology of Kaposi's sarcoma” Cancer Surv 10:5-22, 1991). Therelationship of the disease to geography and immunocompromised patientsled to the suspicion of an infectious agent in Kaposi's sarcomapathogenesis. This suspicion was supported when KSHV or humanherpesvirus 8 (HHV8) was identified through PCR based studies of tumorsamples from AIDS patients with Kaposi's sarcoma (Chang, Y. et al.“Identification of herpesvirus-like DNA sequences in AIDS-associatedKaposi's sarcoma” Science 266:1865-1869, 1994). Subsequent studies haveshown that the virus is of the gammaherpesviridae family, bearingsequence similarity to herpesvirus saimiri (HVS) and Epstein-Barr virus(EBV) (Russo, J. “Nucleotide sequence of the Kaposi sarcoma-associatedherpesvirus (HHV8)” PNAS 93:14862-14867, 1996). Although there isincreasing epidemiologic data associating the virus with human disease,little is known about the biology of this new gammaherpesvirus.

[0004] Indirect immunofluorescence studies of the latently infected BCBLcell line with serum from KS patients reveals a characteristic punctatepattern of nuclear immunofluorescence due to the presence of what wastermed the latency-associated nuclear antigen (LANA) (Moore, P. S., etal. “Primary characterization of a herpesvirus agent with Kaposi'ssarcoma” J. Virol 70:549-558, 1996; Simpson, G. R. et al. “Prevalence ofKaposi's sarcoma associated herpesvirus infection measured by antibodiesto recombinant capsid protein and latent immunofluorescence antigen”Lancet 348:1133-1138, 1996). LANA is detected in the majority of cellsin a KS lesion as well as in cell lines derived from body cavitylymphomas (Simpson, G. R. et al. “Prevalence of Kaposi's sarcomaassociated herpesvirus infection measured by antibodies to recombinantcapsid protein and latent immunofluorescence antigen” Lancet348:1133-1138, 1996; Rainbow, L., et al. “The 222- to 234-kilodaltonlatent nuclear protein (LNA) of Kaposi's sarcoma-associated herpesvirus(human herpes 8) is encoded by orf73 and is a component of thelatency-associated nuclear antigen” J. Virol 71:5915-5921, 1997).Studies based on the detection of antibodies to LANA have shown thatKSHV infection precedes onset of KS and other associatedlymphoproliferative diseases (Gao, S. J., et al. “Seroconversion toantibodies against Kaposi's sarcoma-associated herpesvirus-relatedlatent nuclear antigens before the development of Kaposi's sarcoma” NEngl J Med 335:223-241, 1996). LANA is encoded by orf73 of KSHV and isexpressed as a latency-associated protein in the infected cell (Rainbow,L., et al. “The 222- to 234-kilodalton latent nuclear protein (LNA) ofKaposi's sarcoma-associated herpesvirus (human herpes 8) is encoded byorf73 and is a component of the latency-associated nuclear antigen” J.Virol 71:5915-5921, 1997; Kedes, D. H., et al. “Identification of thegene encoding the major latency-associated nuclear antigen of theKaposi's sarcoma-associated herpesvirus” J Clin Invest 100:2606-2610,1997). An analysis of the LANA amino acid sequence reveals severalacidic and proline/glutamine rich regions as well as a zinc finger DNAbinding domain (Neipel, F., et al. “Fleckenstein Cell-homologous genesin the Kaposi's sarcoma-associated rhadinovirus human herpesvirus 8:determinants of its pathogenicity?” J. Virol 71:4187-4192, 1997; Ganem,D. “KSHV and Kaposi's sarcoma: the end of the beginning of the end?”Cell 91:157-160, 1997). In spite of this suggestion that LANA may act asa transcription factor, a specific function is yet to be assigned forthis viral protein.

[0005] Little is known regarding the mechanism and establishment of KSHVlatency. However, the persistence of the viral genome throughgenerations of host cell divisions potentiates the host's propensity ofcontracting the disease encoded by the virus. What is needed is a drugscreen for agents that would disrupt the ability of a viral genome (e.g.the KSHV genome) to bind to host DNA thereby eliminating the viralgenome in the host.

SUMMARY OF THE INVENTION

[0006] The present invention generally comprises novel compounds andmethods to screen for compounds that interfere with the ability of viralgenomic DNA or RNA to bind to host genomic DNA. Additionally, thepresent invention relates to the targeting of genes to host genomes ingene therapy applications. Furthermore, the present invention relates tocompositions and methods for the treatment of viral infections andtumors.

[0007] Genomic DNA from latent viruses is able to persist for multiplegenerations of the host cell by binding to a tethering protein that isencoded by the viral DNA. We have discovered the mechanism of binding ofthe viral DNA to the host cell. For example, genomic DNA from the Kaposisarcoma virus (KSHV) is able to persist for multiple generations of thehost cell by binding to a tethering protein, the latency-associatednuclear antigen (LANA). LANA tethers the KSHV viral DNA to thechromosomal structural protein, histone 1 (H1). LANA is encoded by theviral DNA, therefore it will only be present in a host cell afterinfection. Likewise, the lack of LANA in a host cell would indicate thelack of viral infection by viruses that utilize this or similar proteinsto ensure persistence. LANA binds to specific locations on the KSHVgenomic DNA designated Z6, Z8 and Z2. We have defined three othersmaller binding regions that partially overlap with Z6, Z8 and Z2, whichwe have named LBR1 (LANA binding region 1), LBR2, LBR3 and LBR4. Theseregions are located at approximately 22-27 kb, 109-111 kb, 127-132 kband a region at the left 1.8 kb of the KSHV genome including one copy ofthe terminal repeat, respectively. LBR1 is located within the Z1 bindingregion, LBR2 is located immediately 3′ to the Z8 binding region and LBR3is located within the Z2 binding region. The Epstein Bar Virus (EBV)persists in host cells by a similar mechanism in that it utilizes atethering protein (EBNA1) to bind the viral genomic DNA to host histoneH1 proteins. These discoveries will permit (among other things) thescreening of agents that interfere with viral DNA binding to host DNA.

[0008] As noted above, the present invention also contemplates screeningassays to identify drugs that inhibit or potentiate the binding oftethering proteins (e.g. LANA and EBNA1) to host histone H1 proteins. Avariety of assay formats are contemplated for testing the potential ofcompounds suspected of modulating tethering protein binding. In oneembodiment, cells are pretreated with the compound suspected ofmodulating the binding of the tethering protein, followed by theaddition of viral DNA or viruses that encode the tethering protein. Acell free assay for the screening of drugs that inhibit or potentiatethe binding of tethering proteins (e.g. LANA and EBNA1) to host histoneH1 proteins is contemplated by the present invention. For example,providing i) histone H1 proteins and ii) LANA or EBNA1; adding acompound or compounds suspected of inhibiting or potentiating thebinding of LANA or EBNA1 to histone H1; and detecting said binding(e.g., by Western blot).

[0009] The invention is not limited to any particular measurementtechnique to measured bound tethering protein. Various methods areenvisioned. In one embodiment, immunofluorescence is used. In anotherembodiment, immunoprecipitation is used. Compounds that inhibittethering protein binding will reveal less bound tethering protein ascompared to controls. Compounds that potentiate the binding of tetheringprotein will reveal increased bound tethering protein as compared tocontrols. The present invention also contemplates the use of highthroughput screening methods. For example, the use of robotics orcomputer controlled systems are contemplated.

[0010] The present invention is not limited to any particular mechanismby which the viral DNA binds to the host cell. For example, the compoundmay inhibit the binding of the viral DNA by competitively inhibitingsaid binding at the binding site, by sequestering the viral DNA, or bysequestering the tethering protein.

[0011] It is not intended that the present invention be limited by thenature of the compounds to be screened in the screening assay of thepresent invention. For example, a variety of compounds includingoligonucleotides, peptides, organic compounds and nonorganic compounds,are contemplated. Additionally, combinations of compounds arecontemplated by the present invention.

[0012] It is not intended that the screening assays of the presentinvention be limited to any particular virus or viral genome. Manydifferent viruses are contemplated to be used in the screening assays.

[0013] It is not intended that the screening assays of the presentinvention be limited to any particular host cell. Many different hostcells are contemplated by the present invention to be used in thescreening assays.

[0014] It is not intended that the screening assays of the presentinvention be limited to any particular tethering protein. Many differenttypes of tethering proteins are contemplated to be use and detected bythe present invention.

[0015] The invention contemplates compounds and methods to be used ingene directed therapy. For example, the invention contemplates the useof the LANA tethering protein (or portion thereof) in conjunction withthe KSHV LANA DNA binding sites for the purpose of targeting DNA thatencodes therapeutic proteins to the chromosomes of the host cell. Theability of LANA to bind histone H1, when used in combination with the agene therapy vector containing a therapeutic protein and the specificKSHV genomic binding regions, will ensure the inclusion and persistenceof the gene sequence in the host cell. It is not intended that thepresent invention be limited to LANA as the tethering protein. The useof other tethering proteins, such as EBNA1, is contemplated.

[0016] The invention also contemplates compounds and methods to be usedin gene directed therapy where the LANA or EBNA1 protein is coupled tothe DNA to be incorporated into the host is bound to the DNA by chemicalmeans. It is not intended that the present invention be limited to aparticular chemical means to bind the LANA protein to the DNA. Forexample, proteins may be ligated to nucleic acids via disulfide bonds(Chu, B. C. and Orgel, L. E. “Ligation of oligonucleotides to nucleicacids or proteins via disulfide bonds” Nucl Acid Res 16:3671-3691,1988). It is not intended that the present invention be limited to anyparticular sequence of DNA to which the tethering protein may bechemically linked. For example, DNA sequences without the KSHV and EBVbinding sites may be used.

[0017] It is not intended that the present invention be limited to aparticular gene therapy. Many different types of gene therapies arecontemplated by the present invention. It is not intended that theinvention be limited to any particular disease. Many diseases areenvisioned as potentially treatable with the present invention. Forexample, multiple sclerosis, Parkinson's disease, Huntington's disease,diabetes and other degenerative diseases are envisioned as beingcandidates for treatment with the present invention.

[0018] It is not intended that the gene therapy of the present inventionbe limited to any particular tethering protein. Many different types oftethering proteins, such as LANA and EBNA1, are contemplated to be usedby the present invention.

[0019] It is not intended that the present invention be limited by theviral DNA binding sites used in the current invention providing thetethering protein recognizes the DNA binding sites.

[0020] The present invention contemplates compounds and methods for thetreatment of viral infections. For example, it is contemplated thatviral vectors can be produced that encode for tethering proteins (e.g.LANA and EBNA1) mutated to bind host histone H1 with greater aviditythan wild type tethering proteins but bind viral DNA binding sites withreduced avidity or do not bind viral DNA binding sites. Such viralvectors would then block access to histone H1 sites thereby preventinginfectious viral DNA from being replicated along with host DNA.

[0021] In one embodiment, the present invention contemplates acomposition comprising a nucleic acid sequence selected from a groupconsisting of SEQ ID NOS.: 8, 11, 12 and 13. In another embodiment, thepresent invention contemplates that the nucleic acid sequence istethered to a nucleic acid binding protein. In another embodiment, thepresent invention contemplates that the tethering is nonreversible. Inyet another embodiment, the present invention contemplates that thenucleic acid binding protein is LANA. In still yet another embodiment,the present invention contemplates that said nucleic acid is virallyderived. In still yet another embodiment, the present inventioncontemplates that the virally derived nucleic is selected from KSHV,EBV, HBV, HCV, HPV, SV40 and HIV viruses.

[0022] In one embodiment, the present invention contemplates a methodfor screening compounds that are agonistic or antagonistic for thetethering of viral proteins to a nucleic acid sequence selected form agroup consisting of SEQ ID NOS.: 8, 11, 12 and 13., said methodcomprising: a) providing; i) a viral DNA binding protein, ii) saidnucleic acid; and, iii) a compound suspected of modulating theinteraction of said viral DNA binding protein with said nucleic acid; b)exposing said viral DNA binding protein and said nucleic acid with saidcompound suspected of being agonistic or antagonistic to the tetheringof said viral DNA binding protein with said nucleic acid; c) assayingfor the presence of said binding. In another embodiment, the presentinvention contemplates that the DNA binding protein is LANA.

DESCRIPTION OF THE FIGURES

[0023]FIG. 1. KSHV DNA is associated with chromosomes in a similarpattern to LANA.

[0024]FIG. 2. LANA preferentially binds specific regions of the KSHVgenome.

[0025]FIG. 3. KSHV DNA colocalizes with LANA to host chromosomes in BC-3cells but not the KSHV negative control BJAB cells.

[0026]FIG. 4. Cis-acting elements within the Z6 and Z2 regions of theKSHV genome and LANA are sufficient to confer colocalization of KSHVviral DNA and LANA to host chromosomes.

[0027]FIG. 5. The KSHV encoded LANA protein interacts with nucleosomalhistone H1 protein.

[0028]FIG. 6. Location of three specific LANA binding regions (LBR)within the KSHV genome.

[0029]FIG. 7. EBNA1 interacts with histone H1 in vitro. H—crude histonesalone; E1—in vitro translated EBNA1 alone; H+E1—crude histones plus invitro translated EBNA1.

[0030]FIG. 8. EBV encoded EBNA1 interacts with histone H1 in EBVinfected LCL1 cells. L-10% lysate not subject to IP; ProA-Protein Abeads preclear; histone H1 IP-immunoprecipitate generated withmonoclonal anti-histone H1 antibody. Non-viral background band appearingin BJAB IP due to nonspecific reaction of polyvalent human sera withcellular antigens.

[0031]FIG. 9. Protein and nucleotide sequences of LANA and EBNA1. Panel(a) shows the nucleotide sequence of LANA (SEQ ID NO: 1). Panel (b)shows the amino acid sequence of LANA (SEQ ID NO:2). Panel (c) shows thenucleotide sequence of EBNA1 (SEQ ID NO:3). Panel (d) shows the aminoacid sequence of EBNA1 (SEQ ID NO:4).

[0032]FIG. 10. Diagrammatic representation of the parent expressionvector used in the present invention.

[0033]FIG. 11. Sequence of the pA3M vector (SEQ ID NO: 22; Invitrogen)used in construction of the LANA plasmid. LANA was cloned into theEco1-EcoRV site. This plasmid is only exemplary. Other plasmids may beused for this invention.

DEFINITIONS

[0034] To facilitate understanding of the invention, a number of termsare defined below.

[0035] As used herein “virus”, “viral” or “virus particle” is used todenote a group of infectious agents characterized by their inability toreproduce outside of a living host cell. Viruses may subvert the hostcell's normal functions, causing the cell to behave in a mannerdetermined by the virus. Likewise, the viral DNA may be “latent”, beingreproduced along with the host DNA without causing undue harm on thehost cell. Additionally, viruses may be constructed in a laboratoryusing recombinant DNA or RNA and preexisting viral coats.

[0036] As used herein “host cell” is used to denote a cell that has beeninfected by one or more virus particles. The virus particles may be wildtype or mutated.

[0037] As used herein “tethering protein” is used to denote a proteinthat functions to attach one or more molecules (e.g. host DNA or hostchromosomal proteins) to one or more other molecules (e.g. expressionvectors for viral DNA). The tethering protein may have other functionsin the cell in addition to functioning as a tethering protein. Theaction of binding one or more molecules by the tethering protein shallbe referred to as “tethering.”

[0038] As used herein “expression vector,” “recombinant DNA vector” or“vector” is used herein to refer to DNA sequences containing a desiredcoding sequence and appropriate DNA sequences necessary for theexpression of the operably linked coding sequence in a particular hostorganism (e.g., mammal). DNA sequences necessary for expression inprocaryotes include a promoter, optionally an operator sequence, aribosome binding site and possibly other sequences. Eukaryotic cells areknown to utilize promoters, polyadenlyation signals and enhancers.

[0039] The phrase “gain-of-function” (gof) as used herein is applicableto the situation where a modified oligonucleotide that, when transfectedinto a host organism and translated into a peptide, results in a peptidethat will function with increased efficiency (e.g. rate of reaction,affinity, etc.) as compared to the wild type peptide. For example, themodified oligonucleotide (or “gof nucleotide”) may, in effect, functionas an augmenter of the natural gene if the natural gene is present andfunctional in the host into which the gof oligonucleotide wastransfected, or it may add that function to the host if the natural geneis not present or is non-functional.

[0040] The phrase “loss-of-function” (lof) as used herein is applicableto the situation where a modified oligonucleotide, when transfected intoa host organism and translated into a peptide, results in a peptide thatfunction with decreased efficiency (e.g. rate of reaction, affinity,etc.) as compared to the wild type peptide. For example, the modifiedoligonucleotide (or “lof” oligonucleotide”) may, in effect, function asa diminisher of natural gene function if the natural gene is present andfunctional in the host into which the lof oligonucleotide wastransfected, or may negatively interfere with processes in the host ifthe natural gene is not present or is non-functional.

[0041] As used herein “agent” or “drug” is used to denote a chemicalcompound (substance), a mixture of chemical compounds, a biologicalmacromolecule, or an extract made from biological materials such asbacteria, plants, fungi, or animal (particularly mammalian) cells ortissues that are suspected of having therapeutic properties. The agentor drug may be purified, substantially purified or partially purified.

[0042] As used herein “agonist” refers to molecules or compounds whichmimic the action of a “native” or “natural” compound. Agonists may behomologous to these natural compounds in respect to conformation, chargeor other characteristics As used herein “antagonist” refers to moleculesor compounds which inhibit the action of a “native” or “natural”compound. Antagonists may or may not be homologous to these naturalcompounds in respect to conformation, charge or other characteristics.

[0043] As used herein, the term “fusion protein” refers to a chimericprotein containing the protein of interest joined to an exogenousprotein fragment. The fusion partner may provide a detectable moiety,may provide an affinity tag to allow purification of the recombinantfusion protein from the host cell, or both. If desired, the fusionprotein may be removed from the protein of interest by a variety ofenzymatic or chemical means known to the art. The present inventioncontemplates portions of LANA and EBNA1 for fusion proteins.

[0044] As used herein, the term “purified” or “to purify” refers to theremoval of contaminants from a sample. The present inventioncontemplates purified compositions (discussed above).

[0045] As used herein, the term “partially purified” refers to theremoval of a moderate portion of the contaminants of a sample to theextent that the substance of interest is recognizable by techniquesknown to those skilled in the art as accounting for an amount of themixture greater than approximately 5% of the total.

[0046] As used herein, the term “substantially purified” refers to theremoval of a significant portion of the contaminants of a sample to theextent that the substance of interest is recognizable by techniquesknown to those skilled in the art as the most abundant substance in themixture.

[0047] As used herein the term “portion” when in reference to a protein(as in “a portion of a given protein”) refers to fragments of thatprotein. The fragments may range in size from four amino acid residuesto the entire amino acid sequence minus one amino acid. In oneembodiment, the present invention contemplates “functional portions” ofa protein. Such portions are “functional” if they contain a bindingregion (i.e. a region having affinity for another molecule) and suchbinding can take place (i.e. the binding region functions, albeit withperhaps lower affinity than that observed for the full-length protein).Such “functional portions” of the gene product are typically greaterthan approximately 10 amino acids in length, and more typically greaterthan approximately 50 amino acids in length, and even more typicallygreater than approximately 100 amino acids in length. “Functionalportions” may also be “conserved portions” of the protein. The alignmentof the various gene products permit one skilled in the art to selectconserved portions of the protein (i.e. those portions in common betweentwo or more species) as well as unconserved portions (i.e. thoseportions unique to two or more species). The present inventioncontemplates conserved portions 10 amino acids in length or greater, andmore typically greater than 50 amino acids in length.

[0048] “Staining” shall be defined as any number of processes known tothose in the field that are used to better visualize, distinguish oridentify a specific component(s) and/or feature(s) of a cell or cells.

[0049] “Immunofluorescence” is a staining technique used to identify,mark, label, visualize or make readily apparent by procedures known tothose practiced in the art, where a ligand (usually an antibody) isbound to a receptor (usually an antigen) and such ligand, if anantibody, is conjugated to a fluorescent molecule, or the ligand is thenbound by an antibody specific for the ligand, and said antibody isconjugated to a fluorescent molecule, where said fluorescent moleculecan be visualized with the appropriate instrument (e.g. a fluorescentmicroscope).

[0050] “Immunoprecipitation” is a technique used to identify, mark,label, visualize or purify (or partially purify) by procedures known tothose practiced in the art, where a ligand (usually an antibody) isbound to a receptor (usually an antigen) and such ligand, if anantibody, is conjugated to a carrier (e.g. protein A-sepharose), or theligand is then bound by an antibody specific for the ligand, and saidantibody is conjugated to a carrier, where the precipitated molecule canthe be released from the antibody, if desired.

[0051] “Antibody” shall be defined as a glycoprotein produced by B cellsthat binds with high specificity to the agent (usually, but not always,a peptide), or a structurally similar agent, that generated itsproduction. Antibodies may be produced by any of the known methodologies[Current Protocols in Immunology (1998) John Wiley and Sons, Inc., N.Y.]and may be either polyclonal or monoclonal.

[0052] “In operable combination”, “in operable order” and “operablylinked” as used herein refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

[0053] “Antigen” shall be defined as a protein, glycoprotein,lipoprotein, lipid or other substance that is reactive with an antibodyspecific for a portion of the molecule.

[0054] “Morphology” shall be defined as the visual appearance of a cellor organism when viewed with the eye, a light microscope, a confocalmicroscope or an electronmicroscope, as appropriate.

[0055] “Patient” shall be defined as a human or other animal, such as aguinea pig or mouse and the like.

[0056] A “wild type” cell or cell line shall be defined as a cell orcell line that retains the characteristics normally associated with thattype of cell or cell line for the physiological process or morphologicalcharacteristic that is being examined. It is permissible for the cell orcell line to have non-wild type characteristics for physiologicalprocess or morphological characteristics that are not being examined aslong as they do not appreciably affect the process or characteristicbeing examined.

[0057] “Tether” and “tethering” (and other grammatical tenses thereof,e.g., tethered) shall be defined as the ability of a compound (e.g. aprotein) to attach (bind) to a first binding partner (e.g. anoligonucleotide) and to a second binding partner (e.g. a secondoligonucleotide or a second protein). Such tethering may be reversibleor nonreversible. In this application, “bind” and “binding” (and othergrammatical tenses thereof, e.g., bound) are synonymous with “tether”and “tethering.”

[0058] “Host” shall be defined as a recipient cell or organism.

[0059] A compound “suspected of modulating the interaction” of two ormore molecules shall be defined as a chemical compound or substance themay increase or decrease binding affinity or avidity of said two or moremolecules, inhibit binding either competitively, noncompetitively orallostericly of said two or more molecules, or alter the temperature,pH, salt concentration or ion concentration at which said two or moremolecules interact.

[0060] “Reversible” binding shall be defined as being capable of througha series of changes in either direction, forward or backward, as, forexample, in a reversible chemical reaction.

[0061] “Irreversible” binding shall be defined as not being capable ofgoing backward through a series of changes. In the present context,irreversible does not mean 100% irreversible but that any reversing ofthe reaction or event is a minor part of the total reaction (i.e., lessthan 5%).

GENERAL DESCRIPTION OF THE INVENTION

[0062] The data present in this application demonstrate that the KSHVgenome is tethered to the host chromosome through its interaction withLANA. Furthermore, it was demonstrated that LANA binds preferentially tocis-acting elements located within the Z2 KSHV cosmid probe and thatchromatin localization depends on the presence of both LANA and Z2.Finally, the immunoprecipitation data shows that LANA binds host histoneH1 in body cavity lymphoma derived cells and in vitro. This interactionhas two potentially important consequences. First, it provides the firstbiochemical link between KSHV episomes and host chromatin that maythereby confer persistence of the KSHV genome in daughter cells. Second,because histone H1 has increasingly been regarded as a modulator ofeukaryotic transcription (Ramakrishnan, V., et al. “Histone structureand the organization of the nucleosome” Annu Rev Biophys Biomol Struct26:83-112, 1997; Wolffe, A. P. “H1” Intl J Biochem and Cellbio29:1463-1466, 1997), LANA may influence expression of H1 transcriptionaltargets through modulation of H1 transcriptional activities. Theseinteractions lead us to assert a model describing a novel mechanism ofviral latency, summarized in FIG. 6. Without limiting the invention toany particular mechanism, in this model, the KSHV encoded LANA tethersthe KSHV episome (Renne, R., et al. “The size and conformation ofKaposi's sarcoma-associated herpesvirus (human herpesvirus 8) DNA ininfected cells and virions” J. Virol 70:8151-8154; Decker, L., et al.“The Kaposi sarcoma-associated herpesvirus (KSHV) is present as anintact latent genome in KS tissue but replicates in the peripheral bloodmononuclear cells of KS patients” J. Exp Med 184:283-288, 1996) to hostchromosomes through interaction with the histone H1 and specificcis-acting elements located within the Z2 region of the genome.

[0063] A synthesis of data collected over several years reveals thatsuch a mechanism of persistent infection is also possible in EBVinfected cells. It is known that EBV episomes (Harris, A., et al.“Random associations of Epstein-Barr virus genomes with host cellmetaphase chromosomes in Burkitt's lymphoma-derived cell lines” J Virol56:328-332, 1985) and EBNA1 (Grogan, E. A., et al. “Two Epstein-Barrviral nuclear neoantigens distinguished by gene transfer, serology andchromosome binding” PNAS 80:7650-7653, 1983) are randomly associatedwith metaphase chromosomes. It has long been thought that EBNA1 wasnecessary for both viral persistence and replication. However, recentstudies suggest that EBNA 1 does not recruit replicative machinery tooriP and may not be required for replication (Aiyar, A., et al. “Theplasmid replicon of EBV consists of multiple cis-acting elements thatfacilitate DNA synthesis by the cell and a viral maintenance element”EMBO 17:63940-6403, 1998). EBNA 1, however, allows persistence of oriPcontaining plasmids. Additionally, histone H1 was identified in aone-hybrid screen for proteins that interact with EBNA 1 bound to oriP(Aiyar, A., et al. “The plasmid replicon of EBV consists of multiplecis-acting elements that facilitate DNA synthesis by the cell and aviral maintenance element” EMBO 17:63940-6403, 1998). In spite of allthese data, colocalization of EBNA1 and EBV to chromosomes as well asthe biochemical basis of their association with chromosomes have notbeen demonstrated.

[0064] Sugden and colleagues hypothesized that EBNA 1 binds to specificDNA sequences on EBV and links this bound DNA through its many linkingdomains by interacting with chromosome associated proteins (Aiyar, A.,et al. “The plasmid replicon of EBV consists of multiple cis-actingelements that facilitate DNA synthesis by the cell and a viralmaintenance element” EMBO 17:63940-6403, 1998). LANA may exhibitanalogous functions to EBNA1 in linking KSHV episomes to hostchromosomes thereby ensuring control of copy number, segregation, andpersistence in the infected cell. LANA may also function as a viraldefense against cellular pathways evolved to eliminate viral geneticmaterial, similar to how EBNA 1 prevents overwhelming episomal loss inEBV infection (Aiyar, A., et al. “The plasmid replicon of EBV consistsof multiple cis-acting elements that facilitate DNA synthesis by thecell and a viral maintenance element” EMBO 17:63940-6403, 1998).

[0065] Additionally, the present invention contemplates that LANA hasspecific binding sites in the viral genome and, although the presentinvention is not limited to specific binding sites, identifies severalviral genome binding sites for LANA.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0066] Generally, the nomenclature used hereafter and the laboratoryprocedures in cell culture, molecular genetics and nucleic acidchemistry and hybridization described below are those well known andcommonly employed in the art. Standard techniques are used forrecombinant nucleic acid methods, polynucleotide synthesis, microbialculture, viral culture and transformation (e.g. electroporation andlipofection). Generally enzymatic reactions and purification steps areperformed according to the manufacturer's specifications. The techniquesand procedures are generally performed according to conventional methodsin the art and various general references (see, generally, Sambrook etal. Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., and Current Protocolsin Molecular Biology (1996) John Wiley and Sons, Inc., N.Y.).

[0067] The present invention contemplates assays for detecting theability of agents to inhibit or enhance the binding of viral DNA to hostchromosomes where high-throughput screening formats are employedtogether with large agent banks (e.g. compound libraries, peptidelibraries and the like) to identify antagonists or agonists. Such viralDNA binding antagonists and agonists may be further developed aspotential therapeutics and diagnostic or prognostic tools for diversetypes of acquired virally transmitted diseases. Additionally, thepresent invention contemplates compounds and methods useful for genetherapy and for the treatment of latent and non-latent viral infections.

[0068] 1. Screens to Identify Agonists and Antagonists of Viral/Host DNABinding Proteins

[0069] There are several different approaches contemplated by thepresent invention to screen for small molecules that specificallyinhibit or enhance the ability of latent viral oligonucleotides to bindto host cells. One approach is to culture the host cells in the presenceof the compound using standard culture procedures, expose the treatedhost cells to the viral particles and then assay for the presence of atethering protein (e.g. LANA or EBNA1) in the host chromosomes usingassays known to those practiced in the art. Cultures of host cells thatwere untreated with the suspected compound or treated with carriersolution would serve as a negative control. The tethering protein couldbe detected in the host cells by, for example, immunofluorescence orwestern blotting. Viral DNA or RNA could be detected in the host cell,for example, by Southern blotting or northern blotting, respectively.

[0070] In another embodiment, a cell-free assay is contemplated. In oneembodiment, this assay comprises, providing i) histone H1, ii) KSHV orEBNA1 protein, and iii) a compound suspected of modulating the bindingof said KSHV or EBNA1 protein to histone H1; a) mixing, in operationalcondition, said histone H1 and said KSHV or EBNA1 protein with saidcompound suspected of modulating the binding of KSHV or EBNA1 to histoneH1 and b) determining the binding of KSHV or EBNA1 to histone H1 by,e.g., western blotting.

[0071] 2. Methods Related to Gene Therapy

[0072] The present invention demonstrates that LANA and EBNA1 aresufficient to tether viral DNA to host cell histone H1 protein.Additionally, the present invention demonstrates that specific regionsof the viral DNA are required for binding of LANA. This technology maybe used in the delivery of therapeutic gene products thereby allowingfor an effective method of gene therapy. In one embodiment, the hostcells are obtained from an immunocompatible donor or from the patient.The host cells are transfected with a construct able to express thetherapeutic protein of interest, (e.g. LANA, EBNA1 or other tetheringprotein) and the viral DNA binding sites for the tethering protein (e.g.Z6, Z2, Z8 or LANA specific consensus sites [LBR1, LBR2, LBR3; seeexperiment 6]). The use of the present invention would permit thetransfected DNA that encodes for the therapeutic protein of interest tobe replicated as the host cells divided, thereby circumventing the majorproblem with gene therapy, the elimination of the transfected DNA by thehost cell. In another embodiment, portions of LANA or EBNA1 arechemically attached to DNA encoding the gene of interest. Morespecifically, for example, the protein may be ligated to a nucleotideencoding a gene of interest via disulfide bonds (Chu, B. C. and Orgel,L. E. “Ligation of oligonucleotides to nucleic acids or proteins viadisulfide bonds” Nucl Acids Res 16:3671-3691, 1988). In yet anotherembodiment, nucleic acid operationally encoding the therapeutic gene ofinterest and a tethering protein such as LANA or EBNA1 may beadministered to a host in absence of the viral protein coat. Details ofthese embodiments follow.

[0073] A. The Multicellular Organism

[0074] Any multicellular organism into which it may be desirable tointroduce exogenous nucleic acid is a potential subject for the presentinvention. The multicellular organism may be a plant or an animal,preferably the latter. The animal is preferably a vertebrate animal, andmore preferably a higher vertebrate, i.e., a mammal or bird, the formerbeing especially preferred. Among mammals, preferred subjects are humanand other primates, laboratory animals such as mice, rats, rabbits andhamsters, pet animals such as dogs and cats, and farm animals such ashorses, cows, goats, pigs and sheep. It will be noted that these animalscome from four orders of class Mammalia: Primata, Rodenta, Carnivora andArtiodactyla.

[0075] B. The Target Cell

[0076] The target cells may belong to tissues (including organs) of theorganism, including cells belonging to (in the case of an animal) itsnervous system (e.g., the brain, spinal cord and peripheral nervouscells), the circulatory system (e.g., the heart, vascular tissue and redand white blood cells), the digestive system (e.g., the stomach andintestines), the respiratory system (e.g., the nose and the lungs), thereproductive system, the endocrine system (the liver, spleen, thyroids,parathyroids), the skin, the muscles, or the connective tissue.

[0077] Alternatively, the cells may be cancer cells derived from anyorgan or tissue of the target organism, or cells of a parasite orpathogen infecting the organism, or virally infected cells of theorganism.

[0078] A useful procedure for hepatic gene therapy requires an efficientand relatively non-invasive approach to the introduction of genes ofinterest into the liver. Several techniques, employing receptor mediatedgene transfer, have been used with some success. However, there is aneed for a readily reproducible procedure which results in the prolongedexpression of the transgene in the liver, even in the absence of partialhepatectomy, and which therefore could be used for human gene therapy.Exogenous DNA has been introduced into hepatocytes of adult animals bytargeting the asialoglycoprotein (ASGP) receptor by means of aligand-poly-L-lysine biconjugate. For the ligand-targeting technique tobe efficient, the DNA must be in a form which permits it to remainintact in the blood and is small enough to be recognized by the ASGPreceptor on the surface of the hepatocytes. Wagner, et al. have targetedgenes to the transferrin receptor in hepatoma cells by condensing theDNA with a poly-L-lysine/transferrin conjugate, into a complex with adiameter of 80-100 nm. This size DNA conjugate is appropriate forrecognition by the transferrin receptor in hepatoma cells, but the ASGPreceptor of hepatocytes discriminates against ligands larger than 10-20nm in diameter.

[0079] A procedure for the introduction of genes into the liver of adultanimals by receptor mediated uptake follows. This procedure haspotential for application to human gene therapy. The major advantages ofthis method are: (i) the ease of preparation of the DNA complex; (ii)the ability to target genes to specific tissues; (iii) the prolongedexpression of the gene in the liver; (iv) the relative safety of thecomplex, since it is devoid of infectious viral DNA; and (v) theepisomal maintenance of the introduced gene.

[0080] C. Targeting

[0081] i. Generally

[0082] In the absence of viral transfer of nucleic acid, compacted DNAmay be targeted to a host cell or cells. “Targeting” is theadministration of the compacted nucleic acid in such a manner that itenters the target cells in amounts effective to achieve the clinicalpurpose. In this regard, it should be noted that DNA and RNA are capableof replication in the nucleus of the target cell, and in consequence theultimate level of the nucleic acid in the cell may increase afteruptake. Moreover, if the clinical effect is mediated by a proteinexpressed by the nucleic acid, it should be noted that the nucleic acidacts as a template, and thus high levels of protein expression can beachieved even if the number of copies of the nucleic acid in the cell islow. Nonetheless, it is desirable to compact high concentrations of DNAto increase the number of target cells which take up the DNA and thenumber of DNA molecules taken up by each cell.

[0083] The route and site of administration may be chosen to enhancetargeting. For example, to target muscle cells, intramuscular injectioninto the muscles of interest would be a logical choice. Lung cells mightbe targeted by administering the compacted DNA in aerosol form. Thevascular endothelial cells could be targeted by coating a ballooncatheter with the compacted DNA and mechanically introducing the DNA.

[0084] In some instances, the nucleic acid binding moiety, whichmaintains the nucleic acid in the compacted state, may also serve as atargeting agent. Polymers of positively charged amino acids are known toact as nuclear localization signals (NLS) in many nuclear proteins.Nonetheless, in some embodiments, targeting may be improved if a targetcell binding moiety is employed.

[0085] ii. Use of Viral Transfer

[0086] The DNA may be packaged in a viral package (i.e. a viral proteincoat encapsulating the DNA). Said viruses may be used to deliver thetherapeutic DNA to the cells of interest. The therapeutic DNA maycontain, in operation condition, a tissue specific promoter to ensurethe expression of the therapeutic gene only in the tissue of interest.The present invention further contemplates the production of retroviralparticles comprising modified (i.e., chimeric) envelope proteinscontaining protein sequences comprising a target binding moiety capableof binding to a receptor. Retrovirus particles bearing these modifiedenvelope proteins may be used to deliver genes of interest to cellsexpressing the receptor. Retroviral particles bearing chimeric proteinscontaining peptide ligands and a portion of the envelope (env) proteinof retroviruses (e.g., ecotropic Moloney murine leukemia virus or avianretroviruses) have been shown to be capable of binding to cellsexpressing the cognate receptor [Kasahara et al. (1994) Science 266:1373and Valsesia-Wittmann et al. (1994) J. Virol. 68:4609].

[0087] iii. Use Of A Target Binding Moiety (TBM)

[0088] If a TBM is used, it must bind specifically to an accessiblestructure (the “receptor”) of the intended target cells. While it is notnecessary that it be absolutely specific for those cells, however, itmust be sufficiently specific for the conjugate to be therapeuticallyeffective. Preferably, its cross-reactivity with other cells is lessthan 20%, more preferably less than 10% and most preferably less than5%.

[0089] There is no absolute minimum affinity which the TBM must have foran accessible structure of the target cell; however, the higher theaffinity, the better. Preferably, the affinity is at least 10³liters/mole, more preferably, at least 10⁶ liters/mole.

[0090] The TBM may be an antibody (or a specifically binding fragment ofan antibody, such as an Fab, Fab, V_(M), V_(L) or CDR[complementarity-determination region]) which binds specifically to anepitope on the surface of the target cell. Methods for raisingantibodies against cells, cell membranes, or isolated cell surfaceantigens are known in the art. Furthermore, the TBM may comprise asingle-chain Fv which binds specifically to an epitope on the surface ofthe target cell. The single-chain Fv may comprise a fusion protein witha NABM (nucleic acid binding moiety) or a therapeutic protein sequence(e.g, an enzyme, cytokine, protein antibiotic, etc.).

[0091] The TBM may be a lectin, for which there is a cognatecarbohydrate structure on the cell surface.

[0092] The target binding moiety may be a ligand which is specificallybound by a receptor carried by the target cells.

[0093] One class of ligands of interest are carbohydrates, especiallymono- and oligosaccharides. Suitable ligands include galactose, lactoseand mannose.

[0094] Another class of ligands of interest are peptides (which hereincludes proteins), such as insulin, epidermal growth factor(s), tumornecrosis factor, prolactin, chorionic gonadotropin, FSH, LH, glucagon,lactoferrin, transferrin, apolipoprotein E, gp120 and albumin.

[0095] The following table lists preferred target binding moieties forvarious classes of target cells: Target Cells Target Binding Moietyliver cells galactose Kupffer cells mannose macrophages mannose lung,liver, intestine Fab fragment vs. polymeric immunoglobulin receptor (pIgR) adipose tissue, insulin lymphocytes Fab fragment vs. CD4 or gp120enterocyte Vitamin B12 muscle insulin fibroblasts mannose-6-phosphatenerve cells Apolipoprotein E

[0096] The target binding moiety may be encompassed with a largerpeptide or protein. The use of a target binding moiety is not strictlynecessary in the case of direct injection of the NA13M/NA condensedcomplex. The target cell in this case is passively accessible to theNABM/NA condensed complex by the injection of the complex to thevicinity of the target cell.

[0097] iv. Liposome-Mediated Gene Transfer

[0098] The possibility of detecting gene expression by encapsulating DNAinto a liposome (body contained by a lipid bilayer) using various lipidand solvent conditions, and injecting the liposome into animal tissues,has been demonstrated. However, despite the potential of this techniquefor a variety of biological systems, the DNA used in these experimentshas not been modified or compacted to improve its survival in the cell,its uptake into the nucleus or its rate of transcription in the nucleusof the target cells. Thus, these procedures have usually resulted inonly transient expression of the gene carried by the liposome.

[0099] Cationic lipids have been successfully used to transfer DNA. Thecationic component of such lipids can compact DNA in solution. Thismethod has been shown to result in heavily aggregated DNA complexesthat, when used for transfecting the DNA in vitro, results in increasedefficiency of gene transfer and expression (relative to naked DNA).Although the formation of these complexes can promote gene transfer invitro, the injection of such complexes in vivo does not result in longlasting and efficient gene transfer.

[0100] A condensation procedure will provide structural features to theDNA/cationic lipid complex that will make it more amenable to prolongedin vivo expression. The combination of such methods could beaccomplished by either of two procedures:

[0101] 1. Formation of condensed DNA complex that is later encapsulatedusing neutral lipids into liposome bodies, or

[0102] 2. Formation of highly condensed unimolecular DNA complexes uponcondensation with cationic lipids could be accomplished. These complexesshould provide a higher efficiency of gene transfer into tissues ofanimals in vivo.

[0103] The procedure of the present invention for the condensation ofDNA, if coupled to the encapsulation of the resulting compacted DNA intoa liposome body, could provide a variety of advantages for transfectioninto animals:

[0104] 1. The liposome promotes the passive fusion with the lipidbilayer of the cytoplasmic membrane of mammalian cells in tissues.

[0105] 2. The condensed DNA could then transfer the genetic informationwith a higher efficiency through the cell compartments to the nucleusfor its expression.

[0106] 3. Condensed DNA could be protected against degradation insidethe cell, thus augmenting the duration of the expression of the newlyintroduced gene.

[0107] 4. Possible immunological response to the polycation condensedDNA could be avoided by the encapsulation with the immunologically inertlipid bilayer.

[0108] D. The Nucleic Acid Binding Moiety

[0109] Any substance which binds reversibly or irreversibly to a nucleicacid may serve as the nucleic acid binding moiety (NABM), provided that(1) it binds sufficiently strongly and specifically to the nucleic acidto retain it until the conjugate reaches and enters the target cell, anddoes not, through its binding, substantially damage or alter the nucleicacid and (2) it reduces the interactions between the nucleic acid andthe solvent, and thereby permits condensation to occur. The ultimatecriterion is one of therapeutic effectiveness of the conjugate.

[0110] Preferably, the NABM is a tethering protein. More preferably, theNAMB is LANA or EBNA1. The NABM may also be a polycation. Its positivelycharged groups bind ionically to the negatively charged DNA, and theresulting charge neutralization reduces DNA-solvent interactions. Apreferred polycation is polylysine. Other potential nucleic acid bindingmoieties include Arg-Lys mixed polymers, polyarginine, polyornithine,histones, avidin, and protamines.

[0111] E. The Nucleic Acid

[0112] Basic procedures for constructing recombinant DNA and RNAmolecules in accordance with the present invention are disclosed bySambrook, J. et al., In: Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989),which reference is herein incorporated by reference.

[0113] The nucleic acid may be a DNA, RNA, or a DNA or RNA derivativesuch as a derivative resistant to degradation in vivo, as discussedbelow. Within this specification, references to DNA apply to othernucleic acids as well, unless clearly forbidden by the context. Thenucleic acid may be single or double stranded. It is preferably of 10 to1,000,000 bases (or base pairs), more preferably 100 to 100,000, and thebases may be same or different. The bases may be the “normal” basesadenine (A), guanine (G), thymidine (T), cytosine (C) and uracil (U), orabnormal bases. The nucleic acid may be prepared by any desiredprocedure.

[0114] In a preferred embodiment, the nucleic acid comprises anexpressible gene which is functional in the target cell, as well as atethering protein (e.g. LANA or EBNA1). Examples of expressible genesmay be, genes that encode coagulation factors, (such as Factor IX),enzymes involved in specific metabolic defects, (such as urea cycleenzymes, especially ornithine transcarbamylase, argininosuccinatesynthase, and carbamyl phosphate synthase); receptors, (e.g., LDLreceptor); toxins; thymidine kinase to ablate specific cells or tissues;ion channels (e.g., chloride channel of cystic fibrosis); membranetransporters (e.g., glucose transporter); and cytoskeletal proteins,(e.g., dystrophin). The gene may be of synthetic, cDNA or genomicorigin, or a combination thereof. The gene may be one which occurs innature, a non-naturally occurring gene which nonetheless encodes anaturally occurring polypeptide, or a gene which encodes a recognizablemutant of such a polypeptide. It may also encode an mRNA which will be“antisense” to a DNA found or an mRNA normally transcribed in the hostcell, but which antisense RNA is not itself translatable into afunctional protein.

[0115] For the gene to be expressible, the coding sequence must beoperably linked to a promoter sequence functional in the target cell.Two DNA sequences (such as a promoter region sequence and a codingsequence) are said to be operably linked if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation in the region sequence to direct thetranscription of the desired gene sequence, or (3) interfere with theability of the gene sequence to be transcribed by the promoter regionsequence. A promoter region would be operably linked to a DNA sequenceif the promoter were capable of effecting transcription of that DNAsequence. In order to be “operably linked” it is not necessary that twosequences be immediately adjacent to one another. A nucleic acidmolecule, such as DNA, is said to be “capable of expressing” a mRNA ifit contains nucleotide sequences which contain transcriptionalregulatory information and such sequences are “operably linked” tonucleotide sequences which encode the RNA. The precise nature of theregulatory regions needed for gene expression may vary from organism toorganism, but in general include a promoter which directs the initiationof RNA transcription. Such regions may include those 5′-non-codingsequences involved with initiation of transcription such as the TATAbox.

[0116] If desired, the non-coding region 3′ to the gene sequence codingfor the desired RNA product may be obtained. This region may be retainedfor its transcriptional termination regulatory sequences, such as thosewhich provide for termination and polyadenylation. Thus, by retainingthe 3′-region naturally contiguous to the coding sequence, thetranscriptional termination signals may be provided. Where thetranscriptional termination signals are not satisfactorily functional inthe expression host cell, then a 3′ region functional in the host cellmay be substituted.

[0117] The promoter may be a “ubiquitous” promoter active in essentiallyall cells of the host organism, e.g., for mammals, the beta-actinpromoter, or it may be a promoter whose expression is more or lessspecific to the target cells. Generally speaking, the latter ispreferred. A promoter native to a gene which is naturally expressed inthe target cell may be used for this purpose, e.g., the PEPCK(phosphoenol pyruvate carboxykinase) promoter for expression inmammalian liver cells. Other suitable promoters include albumin,metallothionein, surfactant, apoe, pyruvate kinase, LDL receptor HMG CoAreductase or any promoter which has been isolated, cloned and shown tohave an appropriate pattern of tissue specific expression and regulationby factors (hormones, diet, heavy metals, etc.) required to control thetranscription of the gene in the target tissue. In addition, a broadvariety of viral promoters can be used; these include MMTV, SV-40 andCMV. An “expression vector” is a vector which (due to the presence ofappropriate transcriptional and/or translational control sequences) iscapable of expressing a DNA (or cDNA) molecule which has been clonedinto the vector and of thereby producing an RNA or protein product.Expression of the cloned sequences occurs when the expression vector isintroduced into an appropriate host cell. If a prokaryotic expressionvector is employed, then the appropriate host cell would be anyprokaryotic cell capable of expressing the cloned sequences. Similarly,when a eukaryotic expression vector is employed, then the appropriatehost cell would be any eukaryotic cell capable of expressing the clonedsequences.

[0118] In addition to or instead of an expressible gene, the nucleicacid may comprise sequences homologous to genetic material of the targetcell, whereby it may insert itself (“integrate”) into the genome byhomologous recombination, thereby displacing a coding or controlsequence of a gene, or deleting a gene altogether.

[0119] In another embodiment, the nucleic acid molecule is “antisense”to a genomic or other DNA sequence of the target organism (includingviruses and other pathogens) or to a messenger RNA transcribed in cellsof the organisms, which hybridizes sufficiently thereto to inhibit thetranscription of the target genomic DNA or the translation of the targetmessenger RNA. The efficiency of such hybridization is a function of thelength and structure of the hybridizing sequences. The longer thesequence and the closer the complementarily to perfection, the strongerthe interaction. As the number of base pair mismatches increases, thehybridization efficiency will fall off. Furthermore, the GC content ofthe packaging sequence DNA or the antisense RNA will also affect thehybridization efficiency due to the additional hydrogen bond present ina GC base pair compared to an AT (or AU) base pair. Thus, a targetsequence richer in GC content is preferable as a target.

[0120] It is desirable to avoid antisense sequences which would formsecondary structure due to intramolecular hybridization, since thiswould render the antisense nucleic acid less active or inactive for itsintended purpose. One of ordinary skill in the art will readilyappreciate whether a sequence has a tendency to form a secondarystructure. Secondary structures may be avoided by selecting a differenttarget sequence.

[0121] An oligonucleotide, between about 15 and about 100 bases inlength and complementary to the target sequence may be synthesized fromnatural mononucleosides or, alternatively, from mononucleosides havingsubstitutions at the non-bridging phosphorous bound oxygens. A preferredanalogue is a methylphosphonate analogue of the naturally occurringmononucleosides. More generally, the mononucleoside analogue is anyanalogue whose use results in oligonucleotides which have the advantagesof (a) an improved ability to diffuse through cell membranes and/or (b)resistance to nuclease digestion within the body of a subject (Miller,P. S. et al., Biochemistry 20:1874-1880 (1981)). Such nucleosideanalogues are well-known in the art. The nucleic acid molecule may be ananalogue of DNA or RNA. The present invention is not limited to use ofany particular DNA or RNA analogue, provided it is capable of fulfillingits therapeutic purpose, has adequate resistance to nucleases, andadequate bioavailability and cell take-up. DNA or RNA may be made moreresistant to in vivo degradation by enzymes, e.g., nucleases, bymodifying internucleoside linkages (e.g., methylphosphonates orphosphorothioates) or by incorporating modified nucleosides (e.g.,2′-O-methylribose or 1′-alpha-anomers). The entire nucleic acid moleculemay be formed of such modified linkages, or only certain portions, suchas the 5′ and 3′ ends, may be so affected, thereby providing resistanceto exonucleases.

[0122] Nucleic acid molecules suitable for use in the present inventionthus include but are not limited to dideoxyribonucleosidemethylphosphonates, see Mill, et al., Biochemistry, 18:5134-43 (1979),oligodeoxynucleotide phosphorothioates, see Matsukura, et al., Proc.Nat. Acad. Sci., 84:7706-10 (1987), oligodeoxynucleotides covalentlylinked to an intercalating agent, see Zerial, et al., Nucleic AcidsRes., 15:9909-19 (1987), oligodeoxynucleotide conjugated withpoly(L-lysine), see Leonetti, et al., Gene, 72:32-33 (1988), andcarbamate-linked oligomers assembled from ribose-derived subunits, seeSummerton, J., Antisense Nucleic Acids Conference, 37:44 (New York1989).

[0123] F. Pharmaceutical Compositions And Methods

[0124] The virally contained nucleic acid, operationally encoding atethering protein such as LANA or EBNA1, may be admixed with apharmaceutically acceptable excipient (i.e., carrier) for administrationto a human or other animal subject. The administration may be by anysuitable route of administration. The dosage form must be appropriatefor that route. Suitable routes of administration and dosage formsinclude intravascular (injectable solution), subcutaneous (injectablesolution, slow-release implant), topical (ointment, salve, cream), andoral (solution, tablet, capsule). With some routes of administration,the dosage form must be formulated to protect the conjugate fromdegradation, e.g., by inclusion of a protective coating or of a nucleaseinhibitor.

[0125] The dosage may be determined by systematic testing of alternativedoses, as is conventional in the art.

[0126] Rats (200-300 g) tolerate as much as 600 μg doses of DNA complexwithout any apparent ill effects on growth or health. Mice (25 g) havebeen injected with 150 μg of that DNA complex without any apparentproblem.

[0127] In humans, a typical trial dose would be 60-120 mg of DNA thismay be increased (or decreased) in a systematic manner, until an optimumdose is identified.

[0128] For short life span cells, e.g., macrophages, a typical dosingschedule might be one dose every two weeks. For long life span cells,e.g., hepatocytes, one dose every two months might be preferable.

[0129] Adjuvants may be used to decrease the size of the DNA complex(e.g., 2-10 mM MgCl), to increase its stability (e.g., sucrose,dextrose, glycerol), or to improve delivery efficiency (e.g.,lysosomotropic agents such as chloroquine and monensine). The complexesmay be enclosed in a liposome to protect them and to facilitate theirentry into the target cell (by fusion of the liposome with the cellmembrane).

[0130] For virally delivered therapeutics, aerosols may be employed.Additionally, the therapeutic viruses may be injected intravenously,subcutaneously, interperatinally or directly into the organ or tissue ofchoice.

[0131] 3. Method Related to the Treatment of Viral Infections

[0132] The present invention contemplates compounds and methods for thetreatment of viral infections. For example, it is contemplated thatviral vectors can be produced that encode for tethering proteins (e.g.LANA and EBNA1) mutated to bind host histone H1 with greater aviditythan wild type but not bind viral DNA binding sites. It is furthercontemplated that the binding of the mutant tethering protein to histoneH1 of the host chromosomes would competitively or noncompetitively blockthe disease causing virus from binding to the host cell and therebyeliminate the persistence of the disease causing virus in the host cell.A virus could be constructed that replaced the native tethering proteinwith the mutant gof (gain-of-function) tethering protein. Such viralvectors would encoded mutant tethering protein that will block histoneH1 sites thereby preventing infectious viral DNA from being replicatedalong with host DNA. After administration of the virus containing themutant tethering protein, the patient would be monitored for progressionof the disease.

[0133] Experimental

[0134] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0135] In the disclosure which follows, the following abbreviationsapply: ° C. (degrees Centigrade); g (gravitational field); vol (volume);w/v (weight to volume); v/v (volume to volume); BSA (bovine serumalbumin); CTAB (cetyltrimethylammonium bromide); fmol (femtomole); FPLC(fast protein liquid chromatography); HEPES(N-[2-Hydroxyethyl]piperazine-N-[2′-ethanesulfonic acid]); HPLC (highpressure liquid chromatography); DTT (dithiothreitol); DMF (N,N dimethylformamide); DNA (deoxyribonucleic acid); i.d. (internal diameter); p(plasmid); μl (microliters); ml (milliliters); μg (micrograms); pmoles(picomoles); mg (milligrams); MOPS (3-[N-Morpholino]propanesulfonicacid); M (molar); mM (milliMolar); μM (microMolar); nm (nanometers);kdal (kilodaltons); OD (optical density); EDTA (ethylene diaminetetra-acetic acid); FITC (fluorescein isothiocyanate); LC sulfo SPDP (LCsulfo-N-succinimidyl-3-(2-pyridyldithio)proprionate); SDS (sodiumdodecyl sulfate); NaPO₄ (sodium phosphate); Tris(tris(hydroxymethyl)-aminomethane); PMSF (phenylmethylsulfonylfluoride);TBE (Tris-Borate-EDTA, e.g., Tris buffer titrated with boric acid ratherthan HCl and containing EDTA); PBS (phosphate buffered saline); PPBS(phosphate buffered saline containing 1 mM PMSF); PAGE (polyacrylamidegel electrophoresis); Tween (polyoxyethylene-sorbitan); BoehringerMannheim or BM (Boehringer Mannheim, Indianapolis, Ind.); New EnglandBiolabs or NEB (New England Biolabs, Beverly, Mass.); Novagen (Novagen,Inc., Madison, Wis.); Pharmacia (Pharmacia Biotech Inc., Piscataway,N.J.); Perkin Elmer (Perkin Elmer, Norwalk, Conn.); Pierce (PierceChemical Co., Rockford, Ill.); Promega (Promega Corp., Madison, Wis.);Qiagen (Qiagen Inc., Chatsworth, Calif.); Stratagene (Stratagene CloningSystems, La Jolla, Calif.); USB or U.S. Biochemical (U.S. Biochemical,Cleveland, Ohio); EMSA (electrophoretic mobility shift assay).

Methods Genomic and cDNA Clones, Cell Culture, and Transfection

[0136] Cosmid and Lambda clones spanning the KSHV genome (Z2, Z6, Z8 andL48, L54, L56, L72, respectively) were obtained from the AIDS Researchand Reference Reagent Program (Russo, J. “Nuclotide sequence of theKaposi sarcoma-associated herpesvirus (HHV8)” Proc Natl Acad Sci93:14862-14867:1996). Orf 73 cDNA was obtained by polymerase chainreaction amplification of DNA using Vent DNA polymerase (NEB) from abody cavity lymphoma derived cell line (BC-1) with the followingprimers: 5′-GAGAATTCTTATGGCGCCCCCGGGAATG-3′ (sense) (SEQ ID NO:5),5′-GAGATATCCCTGTCATTTCCTGTGGAGA-3′ (antisense) (SEQ ID NO:6). Fragmentswere purified, digested with EcoR1 and EcoRV and cloned into amyc-tagged expression vector, pA3M (Aster, J. C., et al. “Oncogenicforms of NOTCH1 lacking either the primary binding site for RBP-Jkappaor nuclear localization sequences retain the ability to associate withRBP-Jkappa and activate transcription” J. Biol. Chem. 272:11336-11343,1997). BC-1, BC-3 (obtained from ATCC), and BJAB (obtained from ElliottKieff) cells were grown in RPMI 1640 (Gibco), supplemented with 20% FCS(10% for BJAB), penicillin (25 U/ml), streptomycin (25 μg/ml), andgentamicin (10 μg/ml). 15 million cells were collected and transfectedwith 50 μg KSHV cosmid DNA (Z2 or Z8) and 50 μg LANA-myc plasmid cDNA(or myc vector alone) by electroporation (220 V, 975 μF). 18 hours aftertransfection, cells were collected for immunoFISH and western blotanalysis.

[0137] FISH analysis. Metaphase chromosome spreads for FISH wereprepared with standard protocols. Briefly, cells were metaphase arrestedwith colcemid (10 μg/ml, Gibco) for 1 hour at 37° C., then treated with0.075 M KCl for 12 minutes at 37° C., followed by overnight fixation infresh methanol/acetic acid (3:1) at 4° C. Cells were spread on slidesand allowed to age for no less than 72 hours. Hybridization was doneovernight at 37° C. with a digoxigenin-labeled KSHV cosmid probe andthen detected with rhodamine conjugated anti-digoxigenin antibodies, andcounterstained with DAPI (4,6-diamidino-2 phenylindole).

[0138] Preparation of Metaphase Chromosomes for ImmunofluorescentAnalysis. Chromosome spreads for immunofluorescence were prepared asabove except that fixation time was shortened to 1 hour to preservechromosome-associated antigens. Slides were blocked in 20% normal goatserum for 30 minutes at room temperature, washed in PBS, and incubatedin human serum reactive to LANA overnight at 4° C. Slides were againwashed and then incubated in goat anti-human FITC secondary antibody(1:1000) for 1 hour at room temperature. Slides were washed,counterstained with DAPI, and coverslipped with antifade solution (2%n-propyl gallate in 70% glycerol) for fluorescence microscopy analysis.

[0139] In vitro DNA binding. Cosmid and lambda probes of KSHV DNA wereradiolabeled with ³²P-dCTP through a standard nick translation protocol(Sambrook, J., et al. “Molecular Cloning: A Laboratory Manual” ColdSpring Harbor Laboratory Press, ed. 2, 1997, pp.3.6-3.12) and separatedfrom unincorporated label with NucTrap probe purification columns(Stratagene). ³⁵S-methionine labeled LANA-myc protein was generated byin vitro translation of a LANA-myc cDNA with rabbit reticulocyte lysate(Promega) as per manufacturer's suggestions. 3 μl of labeled protein wasincubated with 3 μl labeled KSHV probe at 4° C. for 45 minutes in 50 μlEMSA buffer. Simultaneously, 25 μl Protein G-sepharose beads wereincubated with 50 μl monoclonal anti-myc antibody (supernatant from 9E10hybridoma cells) in 350 μl binding buffer rotating at 4° C. for 45minutes. Bound DNA protein complexes were added to prebound Ab-ProteinG-sepharose complexes and incubated for 45 minutes, rotating at 4° C.Complexes were then collected by centrifugation at 15,000 rpm for 1minute at 4° C. in a microcentrifuge. Precipitates were washed twice byremoval of supernatant, resuspension in 200 μl binding buffer, andrecentrifugation. Supernatants from washes were pooled and counted in aliquid scintillation counter as were the corresponding pellets. DNAbound to LANA protein (correcting for amount of protein) was thencalculated with the following formula: (³²P pellet/³⁵S pellet)/(³²Psupernatant/³⁵S supernatant).

[0140] Preparation of metaphase chromosomes for ImmunoFISH doublelabeling. Metaphase chromosome spreads for ImmunoFISH colocalizationwere prepared as above with a 1 hour fixation period to preservechromosome-associated antigens. Hybridization was done overnight at 37°C. with a biotinylated KSHV cosmid or lambda probe and then detectedwith a direct tyramide-rhodamine signal amplification system (NEN LifeSciences), according to manufacturer's suggestions. After repeatedwashes, slides were subjected to the immunofluorescence protocol asabove. Slides were washed, counterstained with DAPI, and coverslippedwith antifade for fluorescence microscopy analysis.

[0141] Immunoprecipitation and in vitro binding. One hundred millionBC-1, BC-3, or BJAB cells were lysed in RIPA buffer and pre-cleared byincubation with protein A alone. Immunoprecipitates were generated withmonoclonal histone H1 antibodies (2 μg/ml-Upstate Biotechnology), washedfour times in RIPA buffer (1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDSand protease inhibitors), then fractionated by SDS-PAGE and transferredto nitrocellulose membranes. Western blots were performed with humanserum reactive to LANA at a 1:500 dilution and standardchemiluminescence detection protocols. For in vitro binding experiments,50 μg of crude histones (type II-A, Sigma) were incubated with 20 μl ofin vitro translated LANA-myc fusion protein for 3 hours at 4° C.Complexes were precipitated by the addition of anti-myc antibodies andprotein G-Sepharose beads followed by centrifugation. Immunoprecipitateswere fractionated by SDS-PAGE and transferred to nitrocellulosemembranes for western blot analysis with monoclonal histone H1antibodies.

[0142] Immunofluorescent colocalization. For colocalization of LANA andhistone H1, slides were blocked in 1% BSA for 30 minutes at roomtemperature, then incubated in 20 μg/ml monoclonal anti-histone H1(Upstate Biotechnology) overnight at 4° C. Slides were washed and thenincubated in anti-LANA serum overnight at 4° C. After more PBS washes,slides were incubated in goat anti-human FITC (1:1000) and donkeyanti-mouse rhodamine (1:100) for 1 hour at room temperature. Slides werewashed in PBS and coverslipped with antifade for analysis on an OlympusAX70 fluorescent microscope.

[0143] Plasmids. The construction of the myc-tagged pA3M LANA expressionplasmid is described elsewhere and is hereby incorporated by reference(Cotter and Robertson, “The Latency-Associated Nuclear Antigen Tethersthe Kaposi's Sarcoma-Associated Herpervirus Genome to Host Chromosomesin Body Cavity-Based Lymphoma Cells” Virology 264:254-264, 1999). Inbrief, the sequences of the present invention were cloned into a versionof the pcDNA3expression vector (Invitrogen, Carlsbad, Calif.; see FIG.10 for the sequence of the parent vector) that was modified to express amyc-tag (Aster, J. C., et al., J. Biol. Chem. 272:11336-11343, 1997).pcDNA3 LANA DC (aa 1-950) and pcDNA3 LANA NDLZ (aa 1-756) were agenerous gift from Dr. Joonhoe Choe, Korea Advanced Institute of Scienceand Technology, Taejeon, Korea (Lim et al., 2000). pA3M LANA 3-9 (aa301-942) and pA3m LANA 7-11 (aa 762-1162) were created by ligation ofthe appropriate PCR products amplified from pA3M LANA into pA3M in framewith the myc epitope tag. pGEX LANA 7-11 (aa 762-1162) was constructedby ligation of a PCR-generated LANA 7-11 cDNA from pA3M LANA into pGEX2TK (Pharmacia) in frame with the N-terminal GST tag. pGL12 was agenerous gift from Dr. Diane Hayward, Johns Hopkins University School ofMedicine, Baltimore, Md. pGL12 represents LANA (aa 940-1177) cloned intothe pGEX-derived pGH413 (Krithivas, et al., J. Virol., 74:9637-9645,2000).

[0144] Cell lines. The 293 pA3MLANA and 293 pA3M vector cell lines havebeen described previously. The KSHV-infected primary effusion lymphomaBC-3 cell line was obtained from ATCC (ATCC catalog number CRL-2277;Arvanitakis, et al., Blood, 88:2648-2654, 1996). The virus-negativeBurkitt's lymphoma cell line BJAB was a gift from Dr. Elliott Kieff,Harvard University, Cambridge, Mass. 293 cells were maintained inDulbecco's modified Eagle medium (Gibco) supplemented with 10% fetalcalf serum, penicillin (25 U/ml), streptomycin (25 mg/ml), andgentamicin (10 mg/ml). BJAB and BC-3 cells were maintained in RPMI 1640(Gibco) supplemented with 10% fetal calf serum (20% for BC-3), and thesame type and concentration of antibiotics as for 293 cells.

[0145] Immunoprecipitation and Western blot analysis. 293 pA3M LANA(50×10⁶) and 293 pA3M (50×10⁶) vector cells were lysed with RIPA buffersupplemented with protease inhibitors as previously described and herebyincorporated by reference (Cotter and Robertson, Virology, 264:254-264,1999). This lysate was precleared with Protein G-Sepharose to eliminatenonspecific binding, followed by the addition of 2 ml monoclonalanti-myc ascites. LANA-myc/anti-myc immune complexes were precipitatedby the addition of 30 ml Protein G-Sepharose followed by centrifugation.Precipitates were washed several times with RIPA buffer and resuspendedin 100 ml EMSA buffer with protease inhibitors. To verify the presenceof LANA in the immunoprecipitate, 10 ml of this sample was subjected toSDS-PAGE and transferred to nitrocellulose for Western blot analysis.The blot was blocked in 6% milk-PBS followed by a 1:300 dilution of themonoclonal anti-myc ascites and a 1:2500 dilution of HRP-conjugatedanti-mouse secondary antibodies (Amersham Pharmacia Biotech.).Alternatively, blots were probed with a 1:50 dilution of serum from a KSpatient (preadsorbed with BJAB extract), followed by a 1:5000 dilutionof a Protein A-HRP secondary (Amersham Pharmacia Biotech.). Standardchemiluminescent procedures (Amersham Pharmacia Biotech.) were used todetect bound antibodies upon exposure to autoradiography film.

[0146] Immunofluorescence. BC-3, BJAB, and 293 LANA or 293 cells werespread on a microscope slide and fixed in acetone/methanol (1:1). Priorto antibody incubation, cells were blocked with 20% normal goat serum,followed by three 5 min washes in 13 PBS. Slides were then incubatedwith a 1:50 dilution of serum from a KS patient preadsorbed with BJABantigens for 1 h at room temperature in a humidity chamber. Slides werewashed three times, 5 min in 13 PBS, prior to incubation withFITC-conjugated goat anti-human secondary antibodies at a dilution of1:1000 in 13 PBS. Slides were again washed four times, 5 min in 13 PBS,prior to mounting in antifade with a coverslip. Slides were examinedusing an Olympus BX60 fluorescence microscope. Images were capturedusing Esprit v1.2 image processing software.

[0147] Randomer-binding experiments. Oligomers (40 bp) with flankingEcoRI and KpnI restrictions were PCR amplified using a synthetictemplate and primers as described previously (Hensel, et al., Science,269:400-403, 1995). Amplified randomers were purified after agarose gelelectrophoresis and resuspended in TE buffer at a concentration of 1mg/ml. Binding reactions were conducted for 30 min at room temperaturein 50 ml EMSA buffer with protease inhibitors. Binding reactionsincluded 10 ml resuspended LANA/Protein G-Sepharose and 2 mg purifiedrandomers. After 30 min LANA/randomer complexes were precipitated bycentrifugation and washed four times in 200 ml EMSA buffer. Washedcomplexes were resuspended in PBS, heated at 65° C. for 20 min, andextracted with phenol:chloroform and then chloroform:isoamyl alcoholbefore ethanol precipitation to recover LANA-binding randomers. Theserecovered randomers were resuspended and used as a template in theaforementioned PCR reaction before again conducting the bindingprotocol. This cycle was completed five consecutive times beforerecovered randomers were digested with EcoRI and cloned into pBluescript(Pharmacia) for sequence analysis. Fifty white transformants wereharvested; DNA was isolated and sequenced with a standard T7 sequencingprimer and the Thermo Sequenase sequencing kit (Amersham PharmaciaBiotech.) according to manufacturers recommendations.

[0148] Protein extracts and electrophoretic mobility shift assay. Probesindicated in Tables 1 and 2 were prepared by using syntheticoligonucleotides (Invitrogen). Complementary oligonucleotides wereannealed and purified from a 12% acrylamide gel in 0.53 TBE by standardprotocols. Probes with GATC 59 overhangs were end-labeled with theKlenow fragment of DNA polymerase I and [³²P]dGTP. Nuclear extracts wereprepared by resuspending 50×10⁶ cells in 500 ml buffer A (10 MM HEPES,10 mM KCl, 1.5 mM MgCl2, 5 mM DTT, 0.5 mM PMSF, 10 mg/ml Aprotinin),followed by a 1-h incubation on ice and then 15 strokes in a Douncehomogenizer. Extracted nuclei were collected by centrifugation andresuspended in 250 ml buffer B (20 mM HEPES, 10% glycerol, 420 mM NaCl,1.5 mM MgCl2, 0.2 mM EDTA, 5 mM DTT, 0.5 mM PMSF, 10 mg/ml Aprotinin).After 30 min on ice, nuclear debris was removed by centrifugation. Thenuclear extract supernatant was added to an equal volume buffer C (20 mMHEPES, 30% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 5 mM DTT, 0.5 mM PMSF,10 mg/ml Aprotinin) and snap frozen in aliquots for storage at 280° C.LANA constructs were transcribed and translated in vitro according tomanufacturer's recommendations (TNT kit, Promega). GST fusion proteinswere prepared after overnight induction of protein expression at 30° C.in LB medium, supplemented with 0.5 mMisopropyl-B-D-thioga-lactopyranoside (IPTG). Bacterial cells wereharvested by centrifugation and resuspended in 1.5 ml NETN (20 mM TrispH 8.0, 0.5% NP-40, 100 mM EDTA, 0.5 mM PMSF, 10 mg/ml Aprotinin). After15 min on ice, 900 ml Sarkosyl in STE (100 mM NaCl, 10 mM Tris pH 7.5, 1mM EDTA) and 75 ml 1 M DTT were added prior to sonication. Cell debriswas removed by centrifugation at 10,000 g for 10 min. The supernatantwas supplemented with 5% Triton X-100 and rotated for 4 h at 4° C. with100 ml glutathione-Sepharose beads (Amersham-Pharmacia Biotech.).Protein concentration for nuclear extracts and purified GST fusionproteins was determined by standard Bradford assay. EMSA-bindingreactions were prepared with either 5 ml protein translated in vitro orapproximately 10 mg nuclear extract, or 1 mg purified GST protein, mixedwith 50 ng deoxyinosine-deoxycytidine (dIdC) nonspecific competitor and20,000 cpm probe in 50 ml buffer: 20 mM HEPES pH 7.5, 0.01% NP-40, 5%glycerol, 10 mM MgCl₂, 100 mg/ml BSA, 2 mM DTT, 1 mM PMSF, 40 mM KCl.For cold competition assays, the indicated amount of cold competitor wasadded 5 min prior to the radiolabeled probe. For supershift assays,monoclonal anti-myc ascites, KS patient serum, rabbit polyclonalanti-LANA, or control mouse IgG (Santa Cruz Biotechnology) was added 5min prior to the probe. Binding reactions proceeded for 15 min at roomtemperature prior to electrophoresis in a 4 or 5% nondenaturingpolyacrylamide gel in 0.53 TBE. After electrophoresis, gels were driedand exposed to autoradiography film or phosphorimager screens (MolecularDevices) for quantitative analysis with ImageQuant software.

Experiment 1

[0149] LANA and KSHV episomes localize similarly to host chromatin inKSHV infected cells. To determine if KSHV achieves latency byintegrating to host DNA, we conducted fluorescent in-situ hybridization(FISH) studies with several viral cosmid probes on metaphase chromosomesprepared from a KSHV positive/EBV negative cell line (BC-3) derived froma body cavity lymphoma as well as a KSHV negative B cell line (BJAB).While viral DNA was not detected in the symmetrical pattern achieved byreplication of a chromatid to which virus has integrated, it wasconsistently detected in a seemingly random association with hostchromosomes (FIG. 1a). Furthermore, when similar chromosome spreadsderived from the same KSHV positive cells were probed with a human serumthat specifically recognizes LANA (FIG. 1b), we demonstrated that LANAlocalized to the host chromosomes in a pattern strikingly similar tothat of KSHV specific DNA hybridization in FIG. 1a. FIG. 1c shows anon-specific control. This chromosome associated pattern ofimmunofluorescence was not evident in metaphase spreads probed withnormal human serum adsorbed against antigens from an EBV negative cellline, BJAB, as well as an EBV positive line, B958 (data not shown),suggesting that the signal is specific for LANA and not due to anonspecific signal from the polyclonal human serum. Therefore ourresults indicated that LANA has the ability to bind sequences on theKSHV genome.

Experiment 2

[0150] LANA displays preferential binding to different regions of KSHVDNA in vitro. To ascertain if LANA had the capacity to bind specificsequences in KSHV DNA, ³²P-dCTP radiolabeled probes spanning the viralgenome (FIG. 2b) were incubated with in vitro translated ³⁵S-methioninelabeled LANA-myc fusion protein, followed by immunoprecipitation withanti-myc antibodies. To detect probes specifically bound to LANA,immunoprecipitates were quantified by liquid scintillation counting.Binding of LANA to DNA was expressed as a percentage of total probe thatcoimmunoprecipitated with LANA-myc. The results of this experiment,shown in FIG. 2a, demonstrated that LANA most preferentially bound aregion of the KSHV genome referred to as Z2, located at approximately127-140 kb on the right end of the viral genome (Russo, J. “Nucleotidesequence of the Kaposi sarcoma-associated herpesvirus (HHV8)” PNAS93:14862-14867, 1996). FIG. 2a is a representative of three separateexperiments in which Z2 bound to LANA-myc more favorably than otherregions of the KSHV genome. Region Z6 at the left end of the viralgenome and L48 bound less preferentially to the immunoprecipitated LANA.Similar to EBNA 1, an EBV protein important for maintenance of EBVepisomes (Mackey, D. and Sugden, B. “Studies on the mechanism of DNAlinking by Epstein-Barr virus nuclear antigen” J. Biol. Chem.272:29873-29879, 1997; Hal Jones, C., et al. “Interaction of thelymphocyte-derived Epstein-Barr virus nuclear antigen EBNA-1 with itsDNA-binding sites” J. Virol. 63:101-110, 1989; Yates, J. L., et al.“Stable replication of plasmids derived from Epstein-Barr virus invarious mammalian cells” Nature 313:812-815, 1985; Rawlins, D. R., etal. “Sequence-specific DNA binding of the Epstein-Barr virus nuclearantigen (EBNA-1) to clustered sites in the plasmid maintenance region”Cell 42:859-868, 1985; Ambinder, R. F., et al. “Definition of thesequence requirements for binding of the EBNA-1 protein to itspalindromic target sites in Epstein-Barr virus DNA” J. Virol.64:2369-2379, 1990), LANA may bind several different sites in the KSHVgenome, suggesting multiple functional roles of LANA in KSHV latentinfection.

Experiment 3

[0151] LANA and KSHV DNA colocalize to metaphase chromosomes in KSHVinfected cells. To determine if LANA colocalized with KSHV DNA,chromosome spreads were generated from cells prepared by fixing for onlyone hour in an effort to retain chromosome-associated antigens thatwould be lost by the typical overnight fixation used in standard FISHprotocols as shown in FIG. 1a. These spreads were probed with KSHV DNAand amplified via a tyramide based fluorochrome deposition(NEN-Lifesciences) for increased sensitivity, followed immediately byanti-LANA immunofluorescence in an effort to colocalize viral DNA andLANA protein at host chromosomes. When both signals from the KSHV probeand the anti-LANA antibody were superimposed it became evident that bothsignals were colocalized to the chromosomes in BC-3 (FIG. 3d) but notBJAB cells (FIG. 3h).

Experiment 4

[0152] Cis-acting DNA elements in Z2 plus LANA are sufficient forchromosomal localization in transfected cells. These data provide thebasis for further experiments in which we tested the hypothesis that Z2,through its preferential binding to LANA, may contain cis-actingelements that can cooperate with LANA to confer chromosome localizationof the KSHV genome. BJAB cells were cotransfected with equivalentamounts of Z2 or Z8 along with an expression construct of a LANA-mycfusion protein under the control of the CMV IE promoter. ImmunoFISH wasthen carried out on these cells, using the appropriate cosmid probefollowed by immunofluorescence with anti-LANA antibodies. Analysis ofthese transfected cells revealed the colocalization of Z2, but not Z8viral DNA with LANA to the host chromosomes (FIG. 4a-c, g-i).Furthermore, this colocalization was dependent on the presence of LANAprotein as Z2 did not localize to host chromosomes when cotransfectedwith empty myc vector (FIG. 4d-f). Interestingly, chromosomes from cellstransfected with Z8 and LANA-myc could not be labeled for either Z8 DNAor LANA protein (FIG. 4g-I), suggesting that the presence of specificviral DNA may be necessary for the stabilization of LANA's interactionwith host chromatin. These data indicate that LANA and cis-actingelements in Z2 are sufficient for chromosome localization of KSHV DNA.Additionally, similar experiments with Z6 demonstrated that Z6 can alsocolocalize with LANA to the host chromosomes (unpublishedobservations-MAC and ESR). Furthermore, our binding studies indicatethat other regions of the KSHV genome (Z6 and L48) may contain othercis-acting elements through which LANA may link the KSHV genome to hostchromosomes.

Experiment 5

[0153] LANA interacts with the nucleosome-associated histone H1 protein.The results of these experiments prompted us to hypothesize that LANAtethers KSHV episomes at Z2 to host chromosomes potentially throughinteraction with chromosomal proteins. Therefore, coimmunoprecipitationexperiments were conducted to determine if LANA specifically interactswith nucleosome-associated histone proteins that could allow for amechanism by which the KSHV genome is tethered to host chromosomes.Immunoprecipitates were generated from BC-1, BC-3, and BJAB cells withantibodies against histone H1 and separated by SDS-PAGE. Western blotanalysis of anti-histone H1 immunoprecipitates using anti-LANA humanserum (FIG. 5a) revealed a 222-234 kD band comigrating in lysate andimmunoprecipitation lanes in BC-I and BC-3 but not in BJAB cells. Asimilar result was generated from the reciprocal experiment. Crudehistones were incubated with in vitro translated LANA-myc fusionprotein, then immunoprecipitated with anti-myc antibodies, andfractionated by SDS-PAGE. Anti-histone H1 western blot analysis of theseimmunoprecipitates revealed a 33 kD comigrating with an identical bandin the histone input lane, but not in control lanes (FIG. 5b).Additionally, through immunofluorescence analysis using antibodiesagainst histone H1 and LANA we have demonstrated that histone H1 andLANA colocalize to metaphase chromosomes in BC-3 cells (FIG. 5c). Giventhe acidic nature of the LANA protein, it was possible that LANA mayalso interact with other chromosomal proteins such as histones H3 andH4, however experiments conducted with specific antibodies to thesenucleosomal antigens (Upstate Biotechnologies) did not yield positiveresults (data not shown).

[0154] Experiment 6

[0155] Localization of three specific LANA binding regions (LBR) withinthe KSHV genome. In order to screen for specific regions of KSHV DNAthat bind DNA, Z2, Z6 and Z8 were pooled and incubated with in vitrotranslated LANA-myc and the immunoprecipitated with monoclonal anti-mycantibodies. These precipitates were washed and then digested withfrequent cutter Sau3A1. These digests were then extracted with phenoland chloroform. Sau3A1 fragments that were protected by interaction withLANA were precipitated from the aqueous phase and ligated into the BamH1site in the MCS of pHbluescript. This ligation was then transformed intocompetent E. coli cells and plated onto LB-ampicillin. Single colonieswere picked for plasmid DNA preparation and insert sequencing. The threeLBR sites, labeled 1, 2 and 3, represent inserts whose sequences showedup redundantly in this experiment and contain sequence similaritieswhich may reflect the presence of a LANA consensus site. These regionsare located at approximately 22-27 kb, 109-111 kb, and 127-132 kb andare shown schematically in FIG. 6.

Experiment 7

[0156] EBNA1 interacts with Histone H1 in vitro. In anothergammaherpesvirus, Epstein-Barr virus (EBV), the protein which is knownto be important for episomal maintenance (EBNA1) also interacts withhistone H1, both in vitro and in EBV infected lymphoblastoid cell lines,suggesting that chromosomal tethering of viral episomes may be aconserved strategy for viral latency. 250 micrograms of crude histoneswere incubated for 1 hour at 4 degrees centigrade with 20 microliters ofin vitro translated EBNA1. Human serum reactive to EBNA1 was added andincubated for one hour at 4 degrees. Immunoprecipitates were generatedby centrifugation after one hour incubation with Protein A-sepharosebeads. After repeated washing, these precipitates were fractionated bySDS-PAGE, transferred to nitrocellulose and probed with a monoclonalanti-histone H1 antibody (FIG. 7).

Experiment 8

[0157] EBV encoded EBNA 1 interacts with histone 1 in EBV infected LCL1cells. 50 million BJAB (non EBV infected) and LCL1 cells were lysed inRIPA buffer and incubated with Protein A-sepharose beads for 1 hour andprecipitated to preclear the lysate of nonspecific interactions (ProAlanes above). After preclear, the lysates were incubated overnight at 4degrees with a monoclonal anti-histone H1 antibody (UpstateBiotechnologies). Immunoprecipitates were generated by the addition ofProtein A-sepharose beads. These precipitates were then SDS-PAGEfractionated, transferred to nitrocellulose and probed with a humanserum reactive to EBNA1 (FIG. 8).

Example 9 Identification of LANA binding Sequences in Viral Genomes

[0158] A. Identification of Potential Consensus LANA-Binding Sites viaRandomer Immunoprecipitation

[0159] In an effort to identify potential LANA-binding sites, a templatefor PCR amplification was synthesized which included KpnI and EcoRIrestriction sites flanking a random 40-bp oligomer. Sense and antisenseprimers, which annealed to the nonrandom KpnI/EcoRI flanking sequences,were used to amplify a large pool of random 40-bp oligomers. This DNAwas purified and mixed with anti-myc immunoprecipitates from 293 cellsstably transduced with a myc-tagged LANA expression construct. After aperiod of 30 min, the immunoprecipitates were centrifuged out of thebinding reaction and washed four times in binding buffer. These beadswere then resuspended, heated at 95° C. for 10 min, and then extractedwith phenol:chloroform followed by chloroform isoamyl alcohol. Oligomersthat had been precipitated with the LANA protein were then recoveredfrom solution by ethanol precipitation. These recovered oligomers werethen used as template in a PCR reaction with the aforementioned primersto enrich the population for oligomers, which contained potentialLANA-binding sites. These amplified DNAs were then subjected to bindingwith immunoprecipitated LANA, recovery, and enrichment by PCRamplification.

[0160] This cycle of PCR amplification followed by LANAimmunoprecipitation was conducted five consecutive times so as togenerate a highly enriched population of oligomers which bound to LANAwith high affinity, while reducing the concentration of oligomers whichmay have bound nonspecifically or with low affinity. This highlyenriched population of oligomers was then digested with EcoRI and clonedinto pBluescript for blue/white screening and sequence analysis ofoligomer inserts. DNA from 50 white ampicillin-resistant colonies wereisolated and sequenced. Sequencing of contained inserts revealedstriking homology among samples. Most inserts fell into one of twocategories, each of which was used to generate a separate consensussequence for alignment with the viral genome. Alignment of thesesequences with the viral genome revealed a similarity between one of theconsensus randomers with a sequence located proximally within the virallong unique region (LUR) at around base pair 125. Closer inspection ofthis region indicated that this sequence was central to an approximately60-bp sequence that retained a dyad symmetry reminiscent of the EBV DSelement. This sequence was used to design a probe for EMSA analysis,terminal LANA-binding region 5 (TLBR5, Table 1). The other consensussequence from the randomer-binding screen aligned with a sequencelocated approximately 1700 bp downstream of the left TR/LUR junction.While not apparently part of a larger DNA structure potentiallyindicative of function in replication, this sequence bore somesimilarity to a sequence which flanks the EBV DS and has been suggestedto serve in the promotion of stability of newly replicated episomes(Yates, et al. J. Virol., 74:4512-4422, 2000). This sequence was alsoused to design a synthetic probe for EMSA analysis, TLBR6 (Table 1).

[0161] B. Identification of Candidate LANA-Binding Sites by Alignment

[0162] Since early DNA-binding screens with LANA revealed the strongestbinding to a restriction fragment from the far left end of the genome,including the terminal repeats, this region was carefully inspected forsequences that might represent part of an origin of replication or otherdiscrete functional elements to which LANA might bind. Given thatorigins of replication in many diverse systems, including latent virusessuch as human papillomavirus (HPV), polyomaviruses, and EBV, containdyad symmetry elements (Lu, et al., J. Virol., 67:7131-7139, 1993;Reisman, et al., Mol. Cell Biol., 5:1822-1832, 1985), we becameinterested in sequences that bore some structural similarities to theseelements. Since LANA has been shown to homodimerize through theC-terminus (Schwam, et al., J. Virol., 74:8532-8540, 2000) and alsoheterodimerize through the C-terminal leucine zipper (Lim, et al., J.Gen. Virol., 81(Pt.11):2645-2652, 2000), we were also interested in anypalindromic or otherwise repeated sequences as it may be reasonablypostulated that functional DNA binding by LANA may involve dimerizationand therefore recruitment of at least two DNA-binding domains. In aneffort to identify this type of cis-acting element, the Megalign(tm)program was employed to align the left end of the genome, including theTR with both its own sense and its antisense strands. An alignment ofthe sense strand with itself would identify direct repeats, whilealignment of the sense with the antisense would reveal palindromicelements or other less obvious regions of dyad symmetry. While thesense/sense alignment did not reveal any regions of significant repeatwithin this region (1TR1 1600-bp LUR), it should be noted thatγ-2-herpesviruses such as KSHV have a relative large number of TR units(˜40). Because of this, any identified site within the TR sequence wouldbe found at least once in each of the 40 TR units. Interestingly, thesense/antisense alignment revealed several regions, which contain eitherpalindromic sequences or some degree of dyad symmetry. Among these wasthe TLBR5 dyad symmetry-like sequence identified in the randomer-bindingscreen. Because of the highly GC-rich nature of the TR, the degree ofalignment in this region is substantially greater than that observed inthe coding LUR. This alignment revealed four sequences other than TLBR5named TLBR1-4 which each displayed more predominantly some degree ofdyad symmetry (Table 1). These sequences were hypothesized to bepotential LANA-binding sites and were used to design EMSA probes to testthis hypothesis. Interestingly, each of these sequences appeared to berelatively unique in that no particular consensus or motif wasidentified as being common among them. TABLE 1 EMSA Probes Used in ThisStudy KSHV Means of Probe Sequence SEQ ID NO coordinates IdentificationLANA Binding TLBR1 atccttgccaatatcctggtatt SEQ ID NO.:9 236-266(LUR^(b)) Megalign Analysis N gcaacaat TLBR2 ttaccgtttggcatctaccaaa SEQID NO.:10 475-505 (LUR^(b)) Megalign Analysis N cggacgaaa TLBR3gcgcgcgcccccccggggg SEQ ID NO.:11 341-371 (TR^(c)) Megalign Analysis Yggtaaaacaggg TLBR4 tcccgcccgggcatggg SEQ ID NO.:12 603-619 (TR^(c))Megalign Analysis Y TLBR5 caatcaagatgttcctgtatgtt SEQ ID NO.:13 97-157(LUR^(b)) Randomer Selection Y gtctgcagtctggcggtttgct ttcgaggactattaagTLBR6 atgaaggatatactctagttgg SEQ ID NO.:14 1683-732 (LUR^(b)) RandomerSelection N accacattccattgcatgtgca gttaat 20bpR^(a) aggagccccggcagcacccSEQ ID NO.:15 24285-305 (LUR^(b)) Genome Inspection N c 30bpR^(b)ctccccggagggggatccc SEQ ID NO.:16 24625-655 (LUR^(b)) Genome InspectionN ggcgcgccacc

[0163] In addition to the TLBR sequences identified at the left end ofthe viral genome, the presence of a highly repetitive region located atabout 24 kb within the LUR was noted. This region spans approximately717 bp between coordinates 24,285 and 24,902 and is apparently devoid ofcoding sequence in the otherwise ORF-rich viral LUR. The region itselfconsists of 17 copies of a 20-bp repeat followed by 9 copies of a 30-bprepeat. Since this structure bears some similarity to the EBV FRsequence of the OriP and was positionally similar to an HVS elementinvolved in maintenance, it was postulated that these repeats mayrepresent a target for LANA binding and perhaps the promotion ofepisomal persistence (Kung and Medveczky, J. Virol., 70:1738-1744,1996). For these reasons each of the two repeat sequences was used todesign EMSA probes to test for binding to LANA (Table 1.).

[0164] C. LANA Specifically Binds to TLBR3, TLBR4, and TLBR5, but notTLBR1, TLBR2, TLBR6 the Repeat Sequences Located at Coordinates 24 kbwithin the KSHV Genome

[0165] To test the identified candidate sequences for binding to LANAprotein, synthetic oligonucleotides for each probe listed in Table 1were annealed with their reverse complement to generate shortdouble-stranded probes for use in an EMSA. The LANA protein wastranslated in vitro along with control luciferase to test for specificbinding to each of the indicated probes. Five microliters of LANA orluciferase translation reaction mixture was mixed with 20,000 cpm ofeach probe (Table 1) for 15 min at room temperature prior toelectrophoresis in a nondenaturing gel and autoradiography. For TLBR3,TLBR4, and TLBR5, there are shifted complexes detected only when invitro translated LANA, but not luciferase, was added to the probe priorto electrophoresis. No specific LANA shift was detected for TLBR1, 2, 6,or either of the repeat sequences shown in Table 1. Each of theLANA-specific shifts detected with TLBR3, 4, and 5 were competed by theaddition of approximately 100-fold excess of cold probe for competition(CC), indicating the specific binding of LANA to each TLBR3, 4, and 5.The same concentration of irrelevant cold probe did not compete.Interestingly, similar LANA-specific shifts were detected with TLBR3 andTLBR5, whereas binding of LANA to TLBR4 resulted in two relativelyslower migrating complexes. It should be noted that these gels were runfor over 18 h to resolve the large complexes and the probes were notresolved along with the specific LANA EMSA shifts.

[0166] D. LANA Binds TLBR4 with Greater Affinity than TLBR3

[0167] To compare the relative affinities of LANA for the threeidentified binding sequences, the ability of each respective probe tocold compete a specific shift was analyzed. In this set of experiments33-, 83-, 165-, 250-, and 500-fold of each cold probe was used tocompete the specific shifts for each of TLBR3, 4, and 5. Neither TLBR3nor TLBR5 were nearly as efficient in specifically competing theLANA/TLBR4 complex as cold TLBR4 itself. In the case of TLBR3, TLBR4appeared to compete with equal efficiency, while TLBR5 competed moreefficiently than TLBR3. In the case of TLBR5, neither TLBR3 nor TLBR4competed as efficiently as TLBR5; however, TLBR4 competed somewhatbetter than TLBR3.

[0168] To get a quantitative indication of the ability of each of theseprobes to compete with one another for binding, analysis using theImageQuant program. By comparing the intensity of the uncompeted LANAcomplex with that of the competed band, a percentage competition can becalculated for each tested concentration for each competitor. Thesenumbers were then used to plot percentage competition (y axis) againstthe fold excess cold competitor (x axis). As suspected, neither TLBR3nor TLBR5 are efficient at competing a specific TLBR4/LANA shift,suggesting that TLBR4 may bind with greater affinity than TLBR5 orTLBR3. In the case of TLBR3, however, TLBR4 is not as efficient as incompeting the TLBR3 shift as TLBR3 or TLBR5. In this case, TLBR5 appearsto compete with as great an efficiency or slightly better than TLBR3.Interestingly, in the case of TLBR5, neither TLBR3 nor TLBR4 ultimatelycompeted with efficiency, approaching that observed for TLBR5. Overall,it appeared that neither TLBR3 nor TLBR5 were capable of completelycompeting a TLBR4/LANA complex; however, the reciprocal experiments,although somewhat less clear, did not show such a distinct modality. Inthese experiments, it appeared that TLBR4 could compete either TLBR3 orTLBR5 with nearly similar efficiency to that observed for cold TLBR3 andTLBR5, respectively. TLBR5 competed TLBR3 with greater specificity thanTLBR4 and was as efficient as TLBR3. This data are consistent with thepossibility that LANA binding to TLBR3 and TLBR5 similarly represents adifferent modality of LANA binding compared to that observed for TLBR4.The EMSA data discussed previously in section C above are alsoconsistent with this possibility in that the TLBR3 and TLBR5/LANAcomplexes migrated similarly, whereas TLBR4/LANA migrated more slowlyand may be representative of a multimer of LANA bound to TLBR4. As TLBR4bound with higher affinity than TLBR3 and TLBR5, we decided to furthercharacterize the binding of TLBR4.

[0169] E. LANA binds TLBR4 and Forms Two Distinct Complexes

[0170] In an effort to better characterize the specificity of LANA forTLBR4 and the constituency of the shifted complexes, the EMSA analysisof TLBR4 with in vitro translated LANA was repeated with a nonspecificDNA control as well as supershift controls. The addition of in vitrotranslated LANA, but not control luciferase (Luc), resulted in thepresence of a high molecular weight complex that migrated as a doublet.Each of these bands are competed by the addition of 1003 excess coldTLBR4 (CC), but not by 1003 excess nonspecific cold DNA (NSCC). Thisdata indicate a high degree of specificity of LANA for the TLBR4sequence. To confirm the presence of LANA in the two shifted bands, amonoclonal anti-myc antibody was added to the binding reaction prior tothe addition of the radiolabeled probe. Since the in vitro translatedLANA protein contains a myc epitope tag, the addition of anti-mycantibody would be expected to result in the presence of supershiftedcomplexes if the shifted bands do indeed reflect LANA binding to theprobe. At least one supershifted complex emerges upon the addition ofthe monoclonal antibody, indicating the presence of LANA in at least thefaster migrating complex. It is possible that the slower band in thedoublet is also supershifted by the antibody; however, this potentialcomplex comigrates with a band that appears in the absence of antibody,rendering its precise identity somewhat unclear. Since probe alone plusthe anti-myc antibody results in neither of the supershifted complexes,the possibility of the antibody binding the probe independent of proteinmay be excluded. Additionally, since control IgG did not result in anysupershifted complex, the possibility of supershifted complexes beingthe result of nonspecific association of antibody with shifted complexesmay also be excluded. These data clearly indicate that LANA binds TLBR4in a sequence-specific fashion.

[0171] F. LANA is Expressed and Localizes to the Nucleus in HEK293 Cells

[0172] To verify appropriate expression of LANA in 293 cells, 15 mg eachof pA3M LANA and pA3M vector alone were transfected by electroporationinto 107 cells. Eighteen hours later cells were harvested and lysed forimmunoblot and immunofluorescence analysis. Lysate from one millioncells from each 293 cell sample was fractionated by SDS-PAGE along withlysates from BJAB uninfected negative control cells as well as BC-3cells, which are latently infected by KSHV and therefore expressendogenous LANA. Fractionated proteins were transferred tonitrocellulose membranes and probed with a KS patient serum reactive toLANA. 293 cells transfected with pA3M LANA express a protein absent inBJAB and vector-transfected cells, which is similar in size toendogenous untagged LANA from BC-3 cells. To confirm the identity of theband detected in the 293 pA3M LANA lysate, the blot was stripped andreprobed with monoclonal anti-myc antibody. The band reactive to the KSserum was also reactive to anti-myc antibodies, confirming its identityas the recombinant myc-tagged LANA expressed from pA3M LANA intransfected 293 cells.

[0173] To verify the nuclear localization of the recombinant LANAexpressed in 293 cells, the same cells analyzed above by immunoblot weresubjected to immunofluorescence analysis using the same KS patientserum, followed by incubation with FITC-conjugated goat anti-humansecondary antibodies. 293 cells transfected with pA3M LANA and BC-3displayed a similar pattern of specific anti-LANA nuclear fluorescence.Very little background fluorescence was detected in vector-transfected293 or uninfected BJAB cells. It was noted that the pattern detected inthe 293 cells was more diffuse and less punctate than that seen in BC-3cells. This has been previously noted and attributed to the absence ofviral episomes to form higher order tethered complexes on the hostchromosomes (Ballestas, et al., Science, 284:641-644, 1999). Theseresults indicate that our pA3MLANA construct expressed LANA, which wastranslocated to the nucleus, similar to that seen with LANA expressedfrom KSHV genome.

[0174] G. Recombinant LANA expressed in 293 cells binds TLBR4

[0175] Since previous binding experiments were conducted with LANAtranslated in vitro, it was of interested whether or not LANA expressedin cells bound to TLBR4 in a similar fashion. To this end nuclearextracts from 293 cells transiently transfected with pA3M LANA andcontrol pA3M vector alone were prepared 18 h after transfection. Theseextracts were used for an EMSA conducted in the same way as with invitro translated LANA. Similar to the results obtained with in vitrotranslated LANA, extracts from 293 cells transfected with the LANA cDNAshifted TLBR4, whereas no such shift was detected when extracts fromvector-transfected 293 cells were added to the probe. The LANA-specificbands were readily competed by the addition of 100-fold excess coldTLBR4, indicating that the presence of the complexes dependsspecifically upon the TLBR4 sequence.

[0176] To verify the presence of LANA in the shifted complexes,supershift analysis was conducted with monoclonal anti-myc antibody(directed against the C-terminal myc epitope tag from pA3M LANA) andcontrol mouse IgG. The addition of anti-myc results in the appearance ofa robust supershifted complex, whereas no such supershifted complex wasetected upon addition of the control mouse IgG. These supershiftedcomplexes are not the result of a nonspecific association betweenantibody and probe, since the addition of anti-myc to probe aloneresulted in no shift whatsoever. This confirms that the shifts detectedfrom 293 LANA cells do in fact contain LANA, perhaps differentiallycomplexed with other cellular proteins. Alternatively the three distinctshifted complexes may reflect different higher order LANA complexes ordifferent posttranslational forms of LANA bound to probe, which maytherefore migrate differently than unmodified LANA.

[0177] H. Endogenous LANA from KSHV-Infected BC-3 Cells Binds TLBR4 inSeveral Distinct Complexes

[0178] To investigate the possibility of endogenous LANA binding to theTLBR4 sequence nuclear extracts from BC-3, a primary effusion lymphomacell line that is latently infected by KSHV and therefore expressesendogenous LANA from resident viral episomes, were generated. Controlextracts were made from BJAB cells, a Burkitt's lymphoma cell line thatis negative for either KSHV or EBV. EMSA analysis was conducted similarto that for the LANA, translated in vitro, and the 293 LANA extracts.Similar results were obtained with the PEL extracts when compared withthat observed for the 293 cells and the LANA translated in vitro. Onceagain, several shifted complexes were detected with the infected BC-3nuclear extracts that were not present upon addition of control BJABextract to the TLBR4 probe. These shifts were also competed with100-fold excess cold TLBR4 (CC), but not with 100-fold excess of anirrelevant probe (NSCC). Interestingly, BC-3 extracts gave rise to atleast six different shifted complexes with TLBR4, suggesting thatperhaps other viral proteins may be recruited to the LANA-containingcomplexes on the TLBR4 sequence. Almost all of the shifts detected withthe BC-3 nuclear extracts were supershifted by the addition of a rabbitpolyclonal anti-LANA antibody, whereas none of these complexes weresupershifted by the addition of the control IgG. This suggests that eachof these complexes does contain LANA and that KSHV-infected cells mayestablish larger heteromeric LANA-containing complexes on this sequencethan extracts that from 293 cells express LANA as the only viralprotein.

[0179] I. Binding to TLBR4 is mediated by the LANA C-terminal domain

[0180] To determine which region of the LANA protein is responsible formediating sequence-specific binding to TLBR4, several truncated LANAconstructs were generated. LANA is a large 1162 amino acid domainprotein with several identified domains. The N-terminus contains aproline-rich (P-rich) domain, while the central portion contains ahighly repetitive acidic region, reminiscent of some transcriptionalactivators. Although the central repeat is highly acidic, the overallamino acid sequence varies. The proximal portion of the repeat, labeledacidic domain (AD), consists primarily of DEED repeats, while theglutamine rich domain (Q-rich) comprises primarily DEQQQ repeats.Variation in the size of this repeat gives rise to some variation in thesize of the LANA protein encoded by different KSHV isolates. ImmediatelyC-terminal to this acidic region lies a hydrophobic heptad repeat regionthat is presumed to form a leucine zipper. With the exception of anidentified nuclear localization signal, the LANA C-terminus does notbear any significant homology to any known protein or domain structure.Based on this domain architecture, four truncated LANA mutant cDNAs werecloned into a eukaryotic expression vector. LANA 1-4 consists of aminoacids 1-435 and includes the N-terminal NLS as well as the proline-richdomain and the first portion of the central repeat. LANA 1-7 consists ofamino acids 1-756 and includes all the sequences in 1-4 plus theglutamine-rich repeat region. LANA 1-950 also includes the leucinezipper and part of the unique C-terminus. LANA 7-11 encodes amino acids762-1162, which represents the entire leucine zipper as well as theentire C-terminus. These constructs were translated in vitro to generatecorresponding LANA polypeptides. These translated LANA proteins areshown in FIG. 8b compared to luciferase (Luc) control.

[0181] To determine which domain mediates DNA binding to TLBR4, each ofthese proteins was subjected to EMSA analysis with the TLBR4 probe. Aspecific shift was detected with the LANA 7-11 polypeptide, but not withany of the other LANA constructs. This shifted complex was readilycompeted with excess cold TLBR4 (CC), but not with a similar excess ofcold irrelevant probe (NSCC), indicating sequence specificity of theLANA 7-11 protein for the TLBR4 sequence. Furthermore, this complex isshown to contain the LANA 7-11 protein as the addition of anti-mycantibodies (directed against the myc epitope tag of LANA 7-11) resultedin a specifically supershifted complex seen before. Control IgG did notgive rise to a supershifted complex nor did anti-myc give rise to anycomplex in the absence of LANA 7-11. These data indicate a specificinteraction between the LANA 7-11 polypeptide and the TLBR4 DNAsequence. Since a LANA polypeptide corresponding to amino acids 1-950did not bind TLBR4, the LANA DNA-binding sequence may be furtherlocalized between amino acids 951 and 1162. The present inventioncontemplates the use of these sequences in diagnostic and therapeuticapplications.

[0182] J. The C-Terminal Leucine Zipper is Not Required for LANA Bindingto TLBR4

[0183] Though these data indicate that the C-terminal 212 amino acidsare necessary for DNA binding, they do not exclude the possibility thatthe leucine zipper is required in addition to the 951-1162 sequence. Totest this possibility, binding of LANA 7-11 was compared to binding of abacterially expressed GST fusion protein that corresponds to the last237 amino acids of LANA (LANA 9-11). This GST protein contains theidentified C-terminal-binding sequence, but lacks the leucine zipperpresent in LANA 7-11. LANA 9-11 gave rise to a similar shift whencompared to that observed for LANA 7-11. As before, this shift wascompeted with excess cold TLBR4, but not by a similar excess of anirrelevant probe of similar length (NSCC). Furthermore this complex wassupershifted by the addition of rabbit anti-LANA antibodies but not withcontrol IgG. The supershift was not as robust as that observed for 7-11;however, this may be due to relatively poor recognition of the LANA 9-11protein by the LANA antibody which was raised against LANA 7-11. Thesedata indicate that binding by LANA of TLBR4 DNA is not dependent on theleucine zipper, but is mediated by the C-terminal 212 amino acids of theLANA protein.

[0184] K. Localization of the Minimal LANA-Binding Sequence within TLBR4

[0185] To identify more precisely the nucleotides critical for LANAbinding to TLBR4, a series of mutated probes were synthesized (Table 2).A close examination of the TLBR4 sequence reveals the presence of an8-bp palindrome (GCCCGGGC [SEQ ID NO.: 7), underlined in Table 2) thatwas postulated to be important for LANA binding. To test thispossibility, three mutant probes were synthesized. TLBR4 HS1 isidentical to TLBR4 except that the first four bases or the firsthalf-site of the palindrome are transverted from GCCC to TAAA (bold,Table 2). TLBR4 HS2 has only the second half-site of the 8-bp palindrometransverted from GGGC to TTTA (bold, Table 2). TLBR4 PD represents onlythe palindrome flanked on either side by one additional nucleotide.

[0186] These probes were subjected to EMSA analysis with the bindingLANA polypeptide, corresponding to the last 400 amino acids (LANA 7-11),translated in vitro. None of these probes gave rise to a specific shiftwith LANA, as compared to the luciferase control. This indicates thateach of the two half-sites of the palindrome are required for binding;however, the 8-bp palindrome itself is insufficient for LANA binding. Tolocalize the additional nucleotides required for binding, two othermutant probes were designed which extended the TLBR4 PD mutant probethree nucleotides in each of the 59 and 39 directions (TLBR4 59, TLBR439, Table 2). These sequences were again tested for binding to LANA viaEMSA. TLBR4 39, but not TLBR4 59, gave rise to a LANA-specific shiftedcomplex. Interestingly, this complex appears to represent relativelyhigh affinity binding as 100-fold excess TLBR4 39 competed the complexmuch less efficiently than the same concentration of cold competitorseen in previous experiments with wild type TLBR4. TLBR4 116 is anotherKSHV sequence located at approximately 116 kb in the KSHV LUR thataligned with the 59 11-bp sequence from TLBR4 located in the KSHV TR,which includes the 8-bp palindrome (Table 2). That LANA did not bindTLBR4 116 suggests that TLBR4 itself repeated in the TR may be the onlyhigh-affinity LANA-binding sequence in the KSHV genome. However, takentogether, these mutagenesis studies indicate that the 13-bp TLBR4 3′sequence (CGCCCGGGCATGG, [SEQ ID NO.: 8]) represents the minimal LANAbinding sequence. TABLE 2 List of TLBR4-Based EMSA Probes Used forMutational Analysis TLBR4-based probes SEQUENCE SEQ ID NO. LANA BindingTLBR4 TCCCGCCCGGGCAT SEQ ID NO.:12 Y GGG TLBR4 HS1 TCCC TAAAGGGCAT SEQID NO.:17 N GGG TLBR4 HS2 TCCCGCCCTTTA ATG SEQ ID NO.:18 N GG TLBR4 PDCGCCCGGGCA SEQ ID NO.:19 N TLBR4 5′ TCCCGCCCGGGCA SEQ ID NO.:20 N TLBR43′ CGCCCGGGCATGG SEQ ID NO.:8 Y TLBR4 116 CGGTCGGTCCCGCC SEQ ID NO.:21 NCGGGGCGCGAA

[0187] From the above it is clear that the present invention providesnovel compounds and methods for the screening of agents that areagonistic or antagonistic for the binding of viral proteins to hostcells. Additionally, the present invention provides novel compounds andmethods useful for gene therapy applications.

1 22 1 3489 DNA Kaposi′s sarcoma-associated herpesvirus 1 atggcgcccccgggaatgcg cctgaggtcg ggacggagca ccggcgcgcc cttaacgaga 60 ggaagttgtaggaaacgaaa caggtctccg gaaagatgtg accttggcga tgacctacat 120 ctacaaccgcgaaggaagca tgtcgccgac tccatcgacg gccgggaatg tggaccccac 180 accttgcctatacctggaag tcccacagtg ttcacatccg ggctgccagc atttgtgtct 240 agtcctactttaccggtggc tcccattcct tcacccgctc ccgcaacacc tttacctcca 300 ccggcactcttaccccccgt aaccacgtct tcctccccaa tccctccatc ccatcctgtg 360 tctccggggaccacggatac tcattctcca tctcctgcat tgccacccac gcagtctcca 420 gagtcttctcaaaggccacc gctttcaagt cctacaggaa ggccagactc ttcaacacct 480 atgcgtccgccaccctcgca gcagactaca cctccacact cacccacgac tcctccaccc 540 gagcctccctccaagtcgtc accagactct ttagctccgt ctaccctgcg tagcctgaga 600 aaaagaaggctatcgtcccc ccaaggtccc tctacactaa acccaatatg tcagtcgccc 660 ccagtctctccccctagatg tgacttcgcc aaccgtagtg tgtacccccc atgggccaca 720 gagtccccgatctacgtggg atcatccagc gatggcgata ctccgccacg ccaaccgcct 780 acatctcccatctccatagg atcatcatcc ccgtctgagg gatcctgggg tgatgacaca 840 gccatgttggtgctccttgc ggagattgca gaagaagcat ccaagaatga aaaagaatgt 900 tccgaaaataatcaggctgg cgaggataat ggggacaacg agattagcaa ggaaagtcag 960 gttgacaaggatgacaatga caataaggat gatgaggagg agcaggagac agatgaggag 1020 gacgaggaggatgacgagga ggatgacgag gaggatgacg aggaggatga cgaggaggat 1080 gacgaggaggatgacgagga ggatgacgag gaggatgacg aggaggatga cgaggaggat 1140 gacgaggaggatgacgagga ggaggacgag gaggaggacg aggaggagga cgaggaggag 1200 gaggacgaggaggatgacga tgatgaggac aatgaggacg aggaggatga cgaggaggag 1260 gacaagaaggaggacgagga ggacgggggc gatggaaaca aaacgttgag catccaaagt 1320 tcacaacagcagcaggagcc acaacagcag gagccacagc agcaggagcc acagcagcag 1380 gagcccctgcaggagccaca acagcaggag ccacagcagc aggagccaca gcagcaggag 1440 cccctgcaggagccacaaca gcaggagcca cagcagcagg agcccctgca ggagccacaa 1500 cagcaggagccacaacagca ggagccacag cagcaggagc cacagcagca ggagccacag 1560 cagcaggagccacagcagca ggagccacag cagcaggagc cacagcagca ggagccacag 1620 cagcaggagccacagcagca ggagccacag cagcgggagc cacagcagcg ggagccccag 1680 cagcgggagccacagcagcg ggagccacag cagcgggagc cacagcagcg ggagccacag 1740 cagcgggagccacagcagcg ggagccacag cagcgggagc cacagcagca ggatgagcag 1800 cagcaggatgagcagcagca ggatgagcag cagcaggatg agcagcagca ggatgagcag 1860 cagcaggatgagcagcagca ggatgagcag cagcaggatg agcagcagca ggatgagcag 1920 cagcaggatgagcagcagca ggatgagcag cagcaggatg agcagcagca ggatgagcag 1980 cagcaggatgagcagcagca ggatgagcag cagcaggatg agcagcagca ggatgagcag 2040 cagcaggatgagcagcagca ggatgagcag cagcaggatg agcagcagca ggatgagcag 2100 gagcagcaggatgagcagga gcagcaggat gagcaggagc agcaggatga gcagcagcag 2160 gatgagcagcagcagcagga tgagcagcag cagcaggatg agcagcagca gcaggatgag 2220 cagcagcagcaggatgagca gcagcagcag gatgaacagg agcagcagga ggagcaggag 2280 cagcaggaggagcaggagca ggagttagag gagcaggagc aggagttaga ggatcaggag 2340 caggagttagaggagcagga gcaggagtta gaggagcagg agcaggagtt agaggagcag 2400 gagcaggagttagaggagca ggagcaggag ttagaggagc aggagcagga gttagaggag 2460 caggagcaggagttagagga gcaggagcag gagttagagg agcaggagca ggagttagag 2520 gagcaggaggtggaagagca agagcaggag gtggaagagc aagagcagga gcaggaagag 2580 caggaattagaggaggtgga ggagcaagag caggagcagg aggagcagga ggagcaggag 2640 ttagaggaggtggaagagca ggaagagcag gagttagagg aggtggaaga gcaggaagag 2700 caggagttagaggaggtgga agagcaggag cagcaggagt tagaggaggt ggaagagcag 2760 gagcagcagggggtggaaca gcaggagcag gagacggtgg aagagcccat aatcttgcac 2820 gggtcgtcatccgaggacga aatggaagtg gattaccctg ttgttagcac acatgaacaa 2880 attgccagtagcccaccagg agataataca ccagacgatg acccacaacc tggcccatct 2940 cgcgaataccgctatgtact cagaacatca ccaccccaca gacctggagt tcgtatgagg 3000 cgcgttccagttacccaccc aaaaaagcca catccaagat accaacaacc accggtccct 3060 tacagacagatagatgattg tcctgcgaaa gctaggccac aacacatctt ttatagacgc 3120 tttttgggaaaggatggaag acgagatcca aagtgtcaat ggaagtttgc agtgattttt 3180 tggggcaatgacccatacgg acttaaaaaa ttatctcagg ccttccagtt tggaggagta 3240 aaggcaggccccgtgtcctg cttgccccac cctggaccag accagtcgcc cataacttat 3300 tgtgtatatgtgtattgtca gaacaaagac acaagtaaga aagtacaaat ggcccgccta 3360 gcctgggaagctagtcaccc cctggcagga aacctacaat cttccatagt taagtttaaa 3420 aagcccctgccattaaccca gccaggggaa aaccaaggtc ctggggactc tccacaggaa 3480 atgacataa3489 2 1162 PRT Kaposi′s sarcoma-associated herpesvirus 2 Met Ala ProPro Gly Met Arg Leu Arg Ser Gly Arg Ser Thr Gly Ala 1 5 10 15 Pro LeuThr Arg Gly Ser Cys Arg Lys Arg Asn Arg Ser Pro Glu Arg 20 25 30 Cys AspLeu Gly Asp Asp Leu His Leu Gln Pro Arg Arg Lys His Val 35 40 45 Ala AspSer Ile Asp Gly Arg Glu Cys Gly Pro His Thr Leu Pro Ile 50 55 60 Pro GlySer Pro Thr Val Phe Thr Ser Gly Leu Pro Ala Phe Val Ser 65 70 75 80 SerPro Thr Leu Pro Val Ala Pro Ile Pro Ser Pro Ala Pro Ala Thr 85 90 95 ProLeu Pro Pro Pro Ala Leu Leu Pro Pro Val Thr Thr Ser Ser Ser 100 105 110Pro Ile Pro Pro Ser His Pro Val Ser Pro Gly Thr Thr Asp Thr His 115 120125 Ser Pro Ser Pro Ala Leu Pro Pro Thr Gln Ser Pro Glu Ser Ser Gln 130135 140 Arg Pro Pro Leu Ser Ser Pro Thr Gly Arg Pro Asp Ser Ser Thr Pro145 150 155 160 Met Arg Pro Pro Pro Ser Gln Gln Thr Thr Pro Pro His SerPro Thr 165 170 175 Thr Pro Pro Pro Glu Pro Pro Ser Lys Ser Ser Pro AspSer Leu Ala 180 185 190 Pro Ser Thr Leu Arg Ser Leu Arg Lys Arg Arg LeuSer Ser Pro Gln 195 200 205 Gly Pro Ser Thr Leu Asn Pro Ile Cys Gln SerPro Pro Val Ser Pro 210 215 220 Pro Arg Cys Asp Phe Ala Asn Arg Ser ValTyr Pro Pro Trp Ala Thr 225 230 235 240 Glu Ser Pro Ile Tyr Val Gly SerSer Ser Asp Gly Asp Thr Pro Pro 245 250 255 Arg Gln Pro Pro Thr Ser ProIle Ser Ile Gly Ser Ser Ser Pro Ser 260 265 270 Glu Gly Ser Trp Gly AspAsp Thr Ala Met Leu Val Leu Leu Ala Glu 275 280 285 Ile Ala Glu Glu AlaSer Lys Asn Glu Lys Glu Cys Ser Glu Asn Asn 290 295 300 Gln Ala Gly GluAsp Asn Gly Asp Asn Glu Ile Ser Lys Glu Ser Gln 305 310 315 320 Val AspLys Asp Asp Asn Asp Asn Lys Asp Asp Glu Glu Glu Gln Glu 325 330 335 ThrAsp Glu Glu Asp Glu Glu Asp Asp Glu Glu Asp Asp Glu Glu Asp 340 345 350Asp Glu Glu Asp Asp Glu Glu Asp Asp Glu Glu Asp Asp Glu Glu Asp 355 360365 Asp Glu Glu Asp Asp Glu Glu Asp Asp Glu Glu Asp Asp Glu Glu Asp 370375 380 Asp Glu Glu Glu Asp Glu Glu Glu Asp Glu Glu Glu Asp Glu Glu Glu385 390 395 400 Glu Asp Glu Glu Asp Asp Asp Asp Glu Asp Asn Glu Asp GluGlu Asp 405 410 415 Asp Glu Glu Glu Asp Lys Lys Glu Asp Glu Glu Asp GlyGly Asp Gly 420 425 430 Asn Lys Thr Leu Ser Ile Gln Ser Ser Gln Gln GlnGln Glu Pro Gln 435 440 445 Gln Gln Glu Pro Gln Gln Gln Glu Pro Gln GlnGln Glu Pro Leu Gln 450 455 460 Glu Pro Gln Gln Gln Glu Pro Gln Gln GlnGlu Pro Gln Gln Gln Glu 465 470 475 480 Pro Leu Gln Glu Pro Gln Gln GlnGlu Pro Gln Gln Gln Glu Pro Leu 485 490 495 Gln Glu Pro Gln Gln Gln GluPro Gln Gln Gln Glu Pro Gln Gln Gln 500 505 510 Glu Pro Gln Gln Gln GluPro Gln Gln Gln Glu Pro Gln Gln Gln Glu 515 520 525 Pro Gln Gln Gln GluPro Gln Gln Gln Glu Pro Gln Gln Gln Glu Pro 530 535 540 Gln Gln Gln GluPro Gln Gln Arg Glu Pro Gln Gln Arg Glu Pro Gln 545 550 555 560 Gln ArgGlu Pro Gln Gln Arg Glu Pro Gln Gln Arg Glu Pro Gln Gln 565 570 575 ArgGlu Pro Gln Gln Arg Glu Pro Gln Gln Arg Glu Pro Gln Gln Arg 580 585 590Glu Pro Gln Gln Gln Asp Glu Gln Gln Gln Asp Glu Gln Gln Gln Asp 595 600605 Glu Gln Gln Gln Asp Glu Gln Gln Gln Asp Glu Gln Gln Gln Asp Glu 610615 620 Gln Gln Gln Asp Glu Gln Gln Gln Asp Glu Gln Gln Gln Asp Glu Gln625 630 635 640 Gln Gln Asp Glu Gln Gln Gln Asp Glu Gln Gln Gln Asp GluGln Gln 645 650 655 Gln Asp Glu Gln Gln Gln Asp Glu Gln Gln Gln Asp GluGln Gln Gln 660 665 670 Asp Glu Gln Gln Gln Asp Glu Gln Gln Gln Asp GluGln Gln Gln Asp 675 680 685 Glu Gln Gln Gln Asp Glu Gln Gln Gln Asp GluGln Glu Gln Gln Asp 690 695 700 Glu Gln Glu Gln Gln Asp Glu Gln Glu GlnGln Asp Glu Gln Gln Gln 705 710 715 720 Asp Glu Gln Gln Gln Gln Asp GluGln Gln Gln Gln Asp Glu Gln Gln 725 730 735 Gln Gln Asp Glu Gln Gln GlnGln Asp Glu Gln Gln Gln Gln Asp Glu 740 745 750 Gln Glu Gln Gln Glu GluGln Glu Gln Gln Glu Glu Gln Glu Gln Glu 755 760 765 Leu Glu Glu Gln GluGln Glu Leu Glu Asp Gln Glu Gln Glu Leu Glu 770 775 780 Glu Gln Glu GlnGlu Leu Glu Glu Gln Glu Gln Glu Leu Glu Glu Gln 785 790 795 800 Glu GlnGlu Leu Glu Glu Gln Glu Gln Glu Leu Glu Glu Gln Glu Gln 805 810 815 GluLeu Glu Glu Gln Glu Gln Glu Leu Glu Glu Gln Glu Gln Glu Leu 820 825 830Glu Glu Gln Glu Gln Glu Leu Glu Glu Gln Glu Val Glu Glu Gln Glu 835 840845 Gln Glu Val Glu Glu Gln Glu Gln Glu Gln Glu Glu Gln Glu Leu Glu 850855 860 Glu Val Glu Glu Gln Glu Gln Glu Gln Glu Glu Gln Glu Glu Gln Glu865 870 875 880 Leu Glu Glu Val Glu Glu Gln Glu Glu Gln Glu Leu Glu GluVal Glu 885 890 895 Glu Gln Glu Glu Gln Glu Leu Glu Glu Val Glu Glu GlnGlu Gln Gln 900 905 910 Glu Leu Glu Glu Val Glu Glu Gln Glu Gln Gln GlyVal Glu Gln Gln 915 920 925 Glu Gln Glu Thr Val Glu Glu Pro Ile Ile LeuHis Gly Ser Ser Ser 930 935 940 Glu Asp Glu Met Glu Val Asp Tyr Pro ValVal Ser Thr His Glu Gln 945 950 955 960 Ile Ala Ser Ser Pro Pro Gly AspAsn Thr Pro Asp Asp Asp Pro Gln 965 970 975 Pro Gly Pro Ser Arg Glu TyrArg Tyr Val Leu Arg Thr Ser Pro Pro 980 985 990 His Arg Pro Gly Val ArgMet Arg Arg Val Pro Val Thr His Pro Lys 995 1000 1005 Lys Pro His ProArg Tyr Gln Gln Pro Pro Val Pro Tyr Arg Gln 1010 1015 1020 Ile Asp AspCys Pro Ala Lys Ala Arg Pro Gln His Ile Phe Tyr 1025 1030 1035 Arg ArgPhe Leu Gly Lys Asp Gly Arg Arg Asp Pro Lys Cys Gln 1040 1045 1050 TrpLys Phe Ala Val Ile Phe Trp Gly Asn Asp Pro Tyr Gly Leu 1055 1060 1065Lys Lys Leu Ser Gln Ala Phe Gln Phe Gly Gly Val Lys Ala Gly 1070 10751080 Pro Val Ser Cys Leu Pro His Pro Gly Pro Asp Gln Ser Pro Ile 10851090 1095 Thr Tyr Cys Val Tyr Val Tyr Cys Gln Asn Lys Asp Thr Ser Lys1100 1105 1110 Lys Val Gln Met Ala Arg Leu Ala Trp Glu Ala Ser His ProLeu 1115 1120 1125 Ala Gly Asn Leu Gln Ser Ser Ile Val Lys Phe Lys LysPro Leu 1130 1135 1140 Pro Leu Thr Gln Pro Gly Glu Asn Gln Gly Pro GlyAsp Ser Pro 1145 1150 1155 Gln Glu Met Thr 1160 3 1926 DNA Epstein-Barrvirus 3 atgtctgacg aggggccagg tacaggacct ggaaatggcc taggagagaagggagacaca 60 tctggaccag aaggctccgg cggcagtgga cctcaaagaa gagggggtgataaccatgga 120 cgaggacggg gaagaggacg aggacgagga ggcggaagac caggagccccgggcggctca 180 ggatcagggc caagacatag agatggtgtc cggagacccc aaaaacgtccaagttgcatt 240 ggctgcaaag ggacccacgg tggaacagga gcaggagcag gagcgggaggggcaggagca 300 ggaggggcag gagcaggagg aggggcagga gcaggaggag gggcaggaggggcaggaggg 360 gcaggagggg caggagcagg aggaggggca ggagcaggag gaggggcaggaggggcagga 420 ggggcaggag caggaggagg ggcaggagca ggaggagggg caggaggggcaggagcagga 480 ggaggggcag gaggggcagg aggggcagga gcaggaggag gggcaggagcaggaggaggg 540 gcaggagggg caggagcagg aggaggggca ggaggggcag gaggggcaggagcaggagga 600 ggggcaggag caggaggggc aggaggggca ggaggggcag gagcaggaggggcaggagca 660 ggaggagggg caggaggggc aggaggggca ggagcaggag gggcaggagcaggaggggca 720 ggagcaggag gggcaggagc aggaggggca ggaggggcag gagcaggaggggcaggaggg 780 gcaggagcag gaggggcagg aggggcagga gcaggaggag gggcaggaggggcaggagca 840 ggaggagggg caggaggggc aggagcagga ggggcaggag gggcaggagcaggaggggca 900 ggaggggcag gagcaggagg ggcaggaggg gcaggagcag gaggaggggcaggagcagga 960 ggggcaggag caggaggtgg aggccggggt cgaggaggca gtggaggccggggtcgagga 1020 ggtagtggag gccggggtcg aggaggtagt ggaggccgcc ggggtagaggacgtgaaaga 1080 gccagggggg gaagtcgtga aagagccagg gggagaggtc gtggacgtggagaaaagagg 1140 cccaggagtc ccagtagtca gtcatcatca tccgggtctc caccgcgcaggccccctcca 1200 ggtagaaggc catttttcca ccctgtaggg gaagccgatt attttgaataccaccaagaa 1260 ggtggcccag atggtgagcc tgacgtgccc ccgggagcga tagagcagggccccgcagat 1320 gacccaggag aaggcccaag cactggaccc cggggtcagg gtgatggaggcaggcgcaaa 1380 aaaggagggt ggtttggaaa gcatcgtggt caaggaggtt ccaacccgaaatttgagaac 1440 attgcagaag gtttaagagc tctcctggct aggagtcacg tagaaaggactaccgacgaa 1500 ggaacttggg tcgccggtgt gttcgtatat ggaggtagta agacctccctttacaaccta 1560 aggcgaggaa ctgcccttgc tattccacaa tgtcgtctta caccattgagtcgtctcccc 1620 tttggaatgg cccctggacc cggcccacaa cctggcccgc taagggagtccattgtctgt 1680 tatttcatgg tctttttaca aactcatata tttgctgagg ttttgaaggatgcgattaag 1740 gaccttgtta tgacaaagcc cgctcctacc tgcaatatca gggtgactgtgtgcagcttt 1800 gacgatggag tagatttgcc tccctggttt ccacctatgg tggaaggggctgccgcggag 1860 ggtgatgacg gagatgacgg agatgaagga ggtgatggag atgagggtgaggaagggcag 1920 gagtga 1926 4 641 PRT Epstein-Barr virus 4 Met Ser AspGlu Gly Pro Gly Thr Gly Pro Gly Asn Gly Leu Gly Glu 1 5 10 15 Lys GlyAsp Thr Ser Gly Pro Glu Gly Ser Gly Gly Ser Gly Pro Gln 20 25 30 Arg ArgGly Gly Asp Asn His Gly Arg Gly Arg Gly Arg Gly Arg Gly 35 40 45 Arg GlyGly Gly Arg Pro Gly Ala Pro Gly Gly Ser Gly Ser Gly Pro 50 55 60 Arg HisArg Asp Gly Val Arg Arg Pro Gln Lys Arg Pro Ser Cys Ile 65 70 75 80 GlyCys Lys Gly Thr His Gly Gly Thr Gly Ala Gly Ala Gly Ala Gly 85 90 95 GlyAla Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly 100 105 110Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly 115 120125 Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala 130135 140 Gly Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly145 150 155 160 Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly GlyAla Gly 165 170 175 Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly GlyAla Gly Gly 180 185 190 Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala Gly AlaGly Gly Ala Gly 195 200 205 Gly Ala Gly Gly Ala Gly Ala Gly Gly Ala GlyAla Gly Gly Gly Ala 210 215 220 Gly Gly Ala Gly Gly Ala Gly Ala Gly GlyAla Gly Ala Gly Gly Ala 225 230 235 240 Gly Ala Gly Gly Ala Gly Ala GlyGly Ala Gly Gly Ala Gly Ala Gly 245 250 255 Gly Ala Gly Gly Ala Gly AlaGly Gly Ala Gly Gly Ala Gly Ala Gly 260 265 270 Gly Gly Ala Gly Gly AlaGly Ala Gly Gly Gly Ala Gly Gly Ala Gly 275 280 285 Ala Gly Gly Ala GlyGly Ala Gly Ala Gly Gly Ala Gly Gly Ala Gly 290 295 300 Ala Gly Gly AlaGly Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala Gly 305 310 315 320 Gly AlaGly Ala Gly Gly Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly 325 330 335 ArgGly Arg Gly Gly Ser Gly Gly Arg Gly Arg Gly Gly Ser Gly Gly 340 345 350Arg Arg Gly Arg Gly Arg Glu Arg Ala Arg Gly Gly Ser Arg Glu Arg 355 360365 Ala Arg Gly Arg Gly Arg Gly Arg Gly Glu Lys Arg Pro Arg Ser Pro 370375 380 Ser Ser Gln Ser Ser Ser Ser Gly Ser Pro Pro Arg Arg Pro Pro Pro385 390 395 400 Gly Arg Arg Pro Phe Phe His Pro Val Gly Glu Ala Asp TyrPhe Glu 405 410 415 Tyr His Gln Glu Gly Gly Pro Asp Gly Glu Pro Asp ValPro Pro Gly 420 425 430 Ala Ile Glu Gln Gly Pro Ala Asp Asp Pro Gly GluGly Pro Ser Thr 435 440 445 Gly Pro Arg Gly Gln Gly Asp Gly Gly Arg ArgLys Lys Gly Gly Trp 450 455 460 Phe Gly Lys His Arg Gly Gln Gly Gly SerAsn Pro Lys Phe Glu Asn 465 470 475 480 Ile Ala Glu Gly Leu Arg Ala LeuLeu Ala Arg Ser His Val Glu Arg 485 490 495 Thr Thr Asp Glu Gly Thr TrpVal Ala Gly Val Phe Val Tyr Gly Gly 500 505 510 Ser Lys Thr Ser Leu TyrAsn Leu Arg Arg Gly Thr Ala Leu Ala Ile 515 520 525 Pro Gln Cys Arg LeuThr Pro Leu Ser Arg Leu Pro Phe Gly Met Ala 530 535 540 Pro Gly Pro GlyPro Gln Pro Gly Pro Leu Arg Glu Ser Ile Val Cys 545 550 555 560 Tyr PheMet Val Phe Leu Gln Thr His Ile Phe Ala Glu Val Leu Lys 565 570 575 AspAla Ile Lys Asp Leu Val Met Thr Lys Pro Ala Pro Thr Cys Asn 580 585 590Ile Arg Val Thr Val Cys Ser Phe Asp Asp Gly Val Asp Leu Pro Pro 595 600605 Trp Phe Pro Pro Met Val Glu Gly Ala Ala Ala Glu Gly Asp Asp Gly 610615 620 Asp Asp Gly Asp Glu Gly Gly Asp Gly Asp Glu Gly Glu Glu Gly Gln625 630 635 640 Glu 5 28 DNA Artificial Sequence Synthetic 5 gagaattcttatggcgcccc cgggaatg 28 6 28 DNA Artificial Sequence Synthetic 6gagatatccc tgtcatttcc tgtggaga 28 7 8 DNA Artificial Sequence Synthetic7 gcccgggc 8 8 13 DNA Artificial Sequence Synthetic 8 cgcccgggca tgg 139 31 DNA Artificial Sequence Synthetic 9 atccttgcca atatcctggtattgcaacaa t 31 10 31 DNA Artificial Sequence Synthetic 10 ttaccgtttggcatctacca aacggacgaa a 31 11 31 DNA Artificial Sequence Synthetic 11gcgcgcgccc ccccgggggg gtaaaacagg g 31 12 17 DNA Artificial SequenceSynthetic 12 tcccgcccgg gcatggg 17 13 61 DNA Artificial SequenceSynthetic 13 caatcaagat gttcctgtat gttgtctgca gtctggcggt ttgctttcgaggactattaa 60 g 61 14 50 DNA Artificial Sequence Synthetic 14 atgaaggatatactctagtt ggaccacatt ccattgcatg tgcagttaat 50 15 20 DNA ArtificialSequence Synthetic 15 aggagccccg gcagcacccc 20 16 30 DNA ArtificialSequence Synthetic 16 ctccccggag ggggatcccg gcgcgccacc 30 17 17 DNAArtificial Sequence Synthetic 17 tccctaaagg gcatggg 17 18 17 DNAArtificial Sequence Synthetic 18 tcccgccctt taatggg 17 19 10 DNAArtificial Sequence Synthetic 19 cgcccgggca 10 20 13 DNA ArtificialSequence Synthetic 20 tcccgcccgg gca 13 21 25 DNA Artificial SequenceSynthetic 21 cggtcggtcc cgcccggggc gcgaa 25 22 5547 DNA ArtificialSequence Synthetic 22 gacggatcgg gagatctccc gatcccctat ggtgcactctcagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgttggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccgacaattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggccagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtcattagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcctggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagtaacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgcccacttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacggtaaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggcagtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaatgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaatgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgccccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctctctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatagggagacccaa gctggctagc 900 gtttaaactt aagcttggta ccgagctcgg atccactagtccagtgtggt ggaattctgc 960 agatatccag cacagtggcg gccgctcgcc accatggaacaaaagctgat ttctgaagaa 1020 gacttggcta gcgaacaaaa gctgatttct gaagaagacttggaacaaaa gctgatttct 1080 gaagaagact tgaccggtta actagctagt ctagagggcccgtttaaacc cgctgatcag 1140 cctcgactgt gccttctagt tgccagccat ctgttgtttgcccctccccc gtgccttcct 1200 tgaccctgga aggtgccact cccactgtcc tttcctaataaaatgaggaa attgcatcgc 1260 attgtctgag taggtgtcat tctattctgg ggggtggggtggggcaggac agcaaggggg 1320 aggattggga agacaatagc aggcatgctg gggatgcggtgggctctatg gcttctgagg 1380 cggaaagaac cagctggggc tctagggggt atccccacgcgccctgtagc ggcgcattaa 1440 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctacacttgccagc gccctagcgc 1500 ccgctccttt cgctttcttc ccttcctttc tcgccacgttcgccggcttt ccccgtcaag 1560 ctctaaatcg ggggctccct ttagggttcc gatttagtgctttacggcac ctcgacccca 1620 aaaaacttga ttagggtgat ggttcacgta gtgggccatcgccctgatag acggtttttc 1680 gccctttgac gttggagtcc acgttcttta atagtggactcttgttccaa actggaacaa 1740 cactcaaccc tatctcggtc tattcttttg atttataagggattttgccg atttcggcct 1800 attggttaaa aaatgagctg atttaacaaa aatttaacgcgaattaattc tgtggaatgt 1860 gtgtcagtta gggtgtggaa agtccccagg ctccccagcaggcagaagta tgcaaagcat 1920 gcatctcaat tagtcagcaa ccaggtgtgg aaagtccccaggctccccag caggcagaag 1980 tatgcaaagc atgcatctca attagtcagc aaccatagtcccgcccctaa ctccgcccat 2040 cccgccccta actccgccca gttccgccca ttctccgccccatggctgac taattttttt 2100 tatttatgca gaggccgagg ccgcctctgc ctctgagctattccagaagt agtgaggagg 2160 cttttttgga ggcctaggct tttgcaaaaa gctcccgggagcttgtatat ccattttcgg 2220 atctgatcaa gagacaggat gaggatcgtt tcgcatgattgaacaagatg gattgcacgc 2280 aggttctccg gccgcttggg tggagaggct attcggctatgactgggcac aacagacaat 2340 cggctgctct gatgccgccg tgttccggct gtcagcgcaggggcgcccgg ttctttttgt 2400 caagaccgac ctgtccggtg ccctgaatga actgcaggacgaggcagcgc ggctatcgtg 2460 gctggccacg acgggcgttc cttgcgcagc tgtgctcgacgttgtcactg aagcgggaag 2520 ggactggctg ctattgggcg aagtgccggg gcaggatctcctgtcatctc accttgctcc 2580 tgccgagaaa gtatccatca tggctgatgc aatgcggcggctgcatacgc ttgatccggc 2640 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgagcgagcacgta ctcggatgga 2700 agccggtctt gtcgatcagg atgatctgga cgaagagcatcaggggctcg cgccagccga 2760 actgttcgcc aggctcaagg cgcgcatgcc cgacggcgaggatctcgtcg tgacccatgg 2820 cgatgcctgc ttgccgaata tcatggtgga aaatggccgcttttctggat tcatcgactg 2880 tggccggctg ggtgtggcgg accgctatca ggacatagcgttggctaccc gtgatattgc 2940 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtgctttacggta tcgccgctcc 3000 cgattcgcag cgcatcgcct tctatcgcct tcttgacgagttcttctgag cgggactctg 3060 gggttcgaaa tgaccgacca agcgacgccc aacctgccatcacgagattt cgattccacc 3120 gccgccttct atgaaaggtt gggcttcgga atcgttttccgggacgccgg ctggatgatc 3180 ctccagcgcg gggatctcat gctggagttc ttcgcccaccccaacttgtt tattgcagct 3240 tataatggtt acaaataaag caatagcatc acaaatttcacaaataaagc atttttttca 3300 ctgcattcta gttgtggttt gtccaaactc atcaatgtatcttatcatgt ctgtataccg 3360 tcgacctcta gctagagctt ggcgtaatca tggtcatagctgtttcctgt gtgaaattgt 3420 tatccgctca caattccaca caacatacga gccggaagcataaagtgtaa agcctggggt 3480 gcctaatgag tgagctaact cacattaatt gcgttgcgctcactgcccgc tttccagtcg 3540 ggaaacctgt cgtgccagct gcattaatga atcggccaacgcgcggggag aggcggtttg 3600 cgtattgggc gctcttccgc ttcctcgctc actgactcgctgcgctcggt cgttcggctg 3660 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggttatccacaga atcaggggat 3720 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaaggccaggaaccg taaaaaggcc 3780 gcgttgctgg cgtttttcca taggctccgc ccccctgacgagcatcacaa aaatcgacgc 3840 tcaagtcaga ggtggcgaaa cccgacagga ctataaagataccaggcgtt tccccctgga 3900 agctccctcg tgcgctctcc tgttccgacc ctgccgcttaccggatacct gtccgccttt 3960 ctcccttcgg gaagcgtggc gctttctcat agctcacgctgtaggtatct cagttcggtg 4020 taggtcgttc gctccaagct gggctgtgtg cacgaaccccccgttcagcc cgaccgctgc 4080 gccttatccg gtaactatcg tcttgagtcc aacccggtaagacacgactt atcgccactg 4140 gcagcagcca ctggtaacag gattagcaga gcgaggtatgtaggcggtgc tacagagttc 4200 ttgaagtggt ggcctaacta cggctacact agaagaacagtatttggtat ctgcgctctg 4260 ctgaagccag ttaccttcgg aaaaagagtt ggtagctcttgatccggcaa acaaaccacc 4320 gctggtagcg gtttttttgt ttgcaagcag cagattacgcgcagaaaaaa aggatctcaa 4380 gaagatcctt tgatcttttc tacggggtct gacgctcagtggaacgaaaa ctcacgttaa 4440 gggattttgg tcatgagatt atcaaaaagg atcttcacctagatcctttt aaattaaaaa 4500 tgaagtttta aatcaatcta aagtatatat gagtaaacttggtctgacag ttaccaatgc 4560 ttaatcagtg aggcacctat ctcagcgatc tgtctatttcgttcatccat agttgcctga 4620 ctccccgtcg tgtagataac tacgatacgg gagggcttaccatctggccc cagtgctgca 4680 atgataccgc gagacccacg ctcaccggct ccagatttatcagcaataaa ccagccagcc 4740 ggaagggccg agcgcagaag tggtcctgca actttatccgcctccatcca gtctattaat 4800 tgttgccggg aagctagagt aagtagttcg ccagttaatagtttgcgcaa cgttgttgcc 4860 attgctacag gcatcgtggt gtcacgctcg tcgtttggtatggcttcatt cagctccggt 4920 tcccaacgat caaggcgagt tacatgatcc cccatgttgtgcaaaaaagc ggttagctcc 4980 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcagtgttatcact catggttatg 5040 gcagcactgc ataattctct tactgtcatg ccatccgtaagatgcttttc tgtgactggt 5100 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggcgaccgagttg ctcttgcccg 5160 gcgtcaatac gggataatac cgcgccacat agcagaactttaaaagtgct catcattgga 5220 aaacgttctt cggggcgaaa actctcaagg atcttaccgctgttgagatc cagttcgatg 5280 taacccactc gtgcacccaa ctgatcttca gcatcttttactttcaccag cgtttctggg 5340 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaataagggcgac acggaaatgt 5400 tgaatactca tactcttcct ttttcaatat tattgaagcatttatcaggg ttattgtctc 5460 atgagcggat acatatttga atgtatttag aaaaataaacaaataggggt tccgcgcaca 5520 tttccccgaa aagtgccacc tgacgtc 5547

1. A composition comprising a nucleic acid sequence selected from agroup consisting of SEQ ID NOS.: 8-21.
 2. The composition of claim 1,wherein said nucleic acid sequence is tethered to a nucleic acid bindingprotein.
 3. The composition of claim 2, wherein said tethering isnonreversible.
 4. The composition of claim 2, wherein said nucleic acidbinding protein is LANA.
 5. The composition of claim 1, wherein saidnucleic acid is virally derived.
 6. The composition of claim 5, whereinsaid virally derived nucleic is selected from KSHV, EBV, HBV, HCV, HPV,SV40 and HIV viruses.
 7. A method for screening compounds that areagonistic or antagonistic for the tethering of viral proteins to anucleic acid sequence selected form a group consisting of SEQ ID NOS.:8, 11, 12 and 13., said method comprising: a) providing; i) a viral DNAbinding protein, ii) said nucleic acid; and, iii) a compound suspectedof modulating the interaction of said viral DNA binding protein withsaid nucleic acid; b) exposing said viral DNA binding protein and saidnucleic acid with said compound suspected of being agonistic orantagonistic to the tethering of said viral DNA binding protein withsaid nucleic acid; c) assaying for the presence of said binding.
 8. Themethod of claim 7 wherein said DNA binding protein is LANA.
 9. Thecomposition of claim 1, wherein said nucleic acid sequence is bound to anucleic acid binding protein.